ANTICANCER RESEARCH 34: 7415-7424 (2014)

Detection of Circulating Tumor Cells in Bladder

Cancer Using Multiplex PCR Assays
IOANNIS LEOTSAKOS1*, PANAGIOTIS DIMOPOULOS2*, ELIONA GKIOKA2,
PAVLOS MSAOUEL2,3, ADRIANOS NEZOS2, KONSTANTINOS G. STRAVODIMOS1,
MICHAEL KOUTSILIERIS2 and CONSTANTINOS A. CONSTANTINIDES1

Departments of 1Urology, and 2Physiology, Medical School,

National & Kapodistian University of Athens, Athens, Greece;
3Department of Internal Medicine, Jacobi Medical Center, Albert Einstein College of Medicine, Bronx, NY, U.S.A.

Abstract. Aim: The aim of this study was to develop

multiplex-PCR assays for the detection of circulating tumor
cells in peripheral blood and urine samples of patients with
bladder cancer. Materials and Methods: Peripheral blood
and urine samples were collected from 208 patients (169
patients and 39 healthy volunteers). After RNA extraction
and cDNA synthesis, the samples were analyzed for the
expression of cytokeratin 19 (CK19), CK20 and epidermal
growth factor receptor (EGFR) mRNA in blood and for
SURVIVIN, human telomerase reverse transcriptase
(hTERT), cytokeratin 20 (CK20) mRNA in urine, using
multiplex-PCR assays. Results: EGFR and CK20 alone or in
combination as well as all urine markers correlated well
with histological grade. hTERT correlated well with primary
tumor size T3. Patients with positive urine markers had
significantly worse progression-free survival. Conclusion:
Multiplex-PCR assays can be a useful tool for staging and
monitoring purposes in patients with bladder cancer.
Bladder cancer is the most common malignancy of the
urinary tract, the eleventh most commonly diagnosed cancer
and a leading cause of cancer morbidity and mortality (1).
The standard procedure for diagnosing bladder cancer
includes cystoscopy combined with cytological examination
(2). Cystoscopy is an invasive, high-cost method and is

This article is freely accessible online.

*These Authors contributed equally to this study.
Correspondence to: Ioannis Leotsakos, Department of Urology,
Medical School, National & Kapodistian University of Athens,
Goudi, Athens, Greece. Tel: +30 2107462597, Fax: +30
2107462571, e-mail: j_leot@yahoo.gr
Key Words: Circulating tumor cells, bladder cancer, PCR, CK19,
CK20, EGFR, SURVIVIN, h-TERT.

0250-7005/2014 $2.00+.40

considered the gold standard for detecting and monitoring

bladder tumors, with a sensitivity of 70%, whereas cytology
is a low-sensitive operator-dependent method (3, 4). There is
a need for sensitive and specic non-invasive urinary
markers that might reduce both cystoscopy and cost. Several
markers are commercially available but none of them alone
is sensitive and specic (5-7). An ideal bladder cancer
marker should have high sensitivity and specificity, it should
be non-invasive and easy to interpret (8).
The presence of circulating tumor cells (CTCs) in the
peripheral blood was first reported by Ashworth in 1869 (9).
Malignant cells are detached from primary tumors and
become invasive. These cells may then intravasate into the
blood or lymphatic circulation and subsequently extravasate
from the circulation and establish a secondary tumor in
another organ far from the primary tumor (10, 11). The
detection of CTCs has been well-demonstrated in breast,
lung, colon, prostate, bladder, melanoma and other
malignancies (12-17). Although CTC detection can
contribute to tumor diagnosis and identify patients with
advanced bladder cancer, such assays cannot be used as
initial screening diagnostic tests due to their low sensitivity
(17). Polymerase chain reaction (PCR)-based methods are
commonly used rapid and low-cost methods for the detection
of CTCs (18).
Cytokeratins (CKs) are intermediate filaments expressed
in epithelial cells. CK20 is more highly expressed in
urothelial tumors compared with normal transitional
epithelium and can, thus, be considered as a marker of
urothelial differentation (19). Epidermal growth factor
receptor (EGFR) is regarded as a specific marker of CTCs
that is mainly correlated with advanced clinical stages (20).
SURVIVIN mRNA detection using reverse transcriptasepolymerase chain reaction (RT-PCR) can be used as an
important adjunct method for cystoscopy in early screening
and postoperative monitoring of bladder cancer (21, 22). In
addition, urinary human telomerase reverse transcriptase

7415

ANTICANCER RESEARCH 34: 7415-7424 (2014)

(hTERT) activity is a good marker for the early diagnosis of
bladder tumors in symptomatic patients (23), having the
same specificity as urinary bladder cytology but higher
sensitivity (24), while the combination of hTERT with
cytology increases sensitivity to 95% (25).
The aim of this study was the development of a multiplexPCR assay for the detection of CTCs in patients with bladder
cancer using primers for CK19, CK20, EGFR in peripheral
blood, and SURVIVIN, hTERT and CK20 in urine samples,
and to assess its potential use for clinical applications.

Materials and Methods

Study participants. A total of 208 patients (177 men and 31 women)
from one tertiary University Hospital, prior to surgical or any other
therapeutic intervention were enrolled in the study. They were
categorized into three groups. Group one included patients newlydiagnosed with bladder cancer (n=105), group two included patients
with history of bladder cancer (n=64), and group three included
healthy volunteers (n=39). People without clinical or laboratory
suspicion, without a known history of malignancy or who were
operated for another reason entirely irrelevant (e.g. inguinal hernia),
considered as healthy volunteers. Patients were excluded from the
study if they had another histological type of cancer, had received
neoadjuvant or adjunant chemotherapy, and had a medical history
of the same or another type of cancer.
In the group of patients with a history of bladder cancer, less than
five years had passed from the last occurrence, without any
recurrence noted in the interim. Samples in this group were
collected before their admission to hospital either for regular followup or early workup due to suspicion of recurrence. Clinical staging
was based on pathological findings at the time of transurethral
resection, examination under anesthesia, cross-section imaging of
the abdomen and pelvis with either computed tomography or
magnetic resonance imaging, and chest X-ray. Transurethral
resection and radical cystectomy specimens were graded and staged
according to TNM classification. Patients were also categorized
based on the American Joint Committee on Cancer (AJCC) staging.
The clinical and demographic characteristics of the patients are
shown in Table I. All participants provided written informed consent
to this research protocol and this study was conducted under the
approval of the local Ethics Committee (Approval number of
scientific council: E.S. 55/07-04-2011) and conforms to the
Declaration of Helsinki.
Primer design. Primers for the selected markers were designed
using FastPCR software (http://primerdigital.com/fastpcr.html). The
sequences of the five primer pairs were additionally tested on NCBI
Nucleotide Blast (http://blast.ncbi.nlm.nih.gov/blast) to confirm that
they were specific for each target. Glyceraldehyde-3-phosphate
dehydro-genase (GAPDH) was used to evaluate the performance of
cDNA synthesis (housekeeping gene). The sequences of the assay
primers are presented in Table II.
RNA isolation. Six milliliters of blood were drawn from each
participant, using a venous catheter, into 3 ml EDTA-containing
vacutainers. Fifty milliliters of spontaneously voided urine (second
void of the day) was taken from each patient before they received
any treatment or underwent surgery.

7416

Table I. Clinical and demographic characteristics for the three study

groups.
Group

Gender
Men
Women
Mean age (SD), years
Primary tumor stage (T)
0a
0is
1
2
3
4
Infiltrated Nodes (N)
No
Yes
Metastasis (M)
No
Yes
Anatomical stage*
0a
0is
1
2
3
4
Histological grade
1
2
3
In situ

Newlydiagnosed
(n=105)

History of
bladder
Ca (N=64)

Control
(n=39)

n (%)

n (%)

n (%)

90 (85.7)
15 (14.3)
68.4 (10.5)

58 (90.6)
6 (9.4)
68.5 (10.9)

36
1
34
20
9
5

20
4
34
4
1
1

(34.3)
(1)
(32.4)
(19)
(8.6)
(4.8)

29 (74.4)
10 (25.6)
67.2 (18.0)

(31.3)
(6.3)
(53.1)
(6.3)
(1.6)
(1.6)

97 (92.4)
8 (7.6)

62 (96.9)
2 (3.1)

105 (100)
0 (0)

64 (100)
0 (0)

35
3
33
16
7
11

(33.3)
(2.9)
(31.4)
(15.2)
(6.7)
(10.5)

20
4
34
3
1
2

(31.3)
(6.3)
(53.1)
(4.7)
(1.6)
(3.1)

8
46
50
1

(7.6)
(43.8)
(47.6)
(1)

5
29
26
4

(7.8)
(45.3)
(40.6)
(6.3)

*By American Joint Committee on Cancer (50).

Blood and urine samples were processed within 3 h of collection.

Erythrocyte Lysis Buffer (ELB) (155 mM NH4Cl, 10 mM KHCO3
and 0.1 mM EDTA, pH of 7.4) was used to lyse all erythrocytes by
osmosis and enrich the samples in nucleated leucocytes. ELB (7.5
ml) was added to each sample and the samples were kept on ice for
45 minutes, with occasional mixing by inversion and they were then
centrifuged at 400g for 10 min at 4C. The supernatant was discarded
and if red blood cells still remained in the sample, 5 ml of ELB were
added to the pellet, the cells were resuspended, kept on ice for an
additional 10 min and the centrifugation step was repeated. Urine
samples were centrifuged at 2000 g for 10 min. The cell pellet was
homogenized in 1 ml Tri-Reagent RT-111 (MRC Inc., Cincinnati,
OH,USA). Total cellular RNA was then extracted, according to the
manufacturers instructions. Diethylpyrocarbonate water (DEPCtreated H2O) was used for RNA pellet dilution. Total RNA
concentration and quality were determined by ultraviolet
spectophotometry (measurements at 260 nm, 280 nm).
cDNA synthesis. cDNA was synthesized using Moloney Murine

Leotsakos et al: CTCs in Bladder Cancer Using Multiplex PCR

Table II. Sequences of the assay primers.

Gene

Gene name

Product size

Sequence

CK19

Cytokeratin 19

159 bp

CK20

Cytokeratin 20

371 bp

EGFR

Epidermal growth factor receptor

480 bp

SURVIVIN

258 bp

hTERT

Baculoviral inhibitor of apoptosis

Repeat-containing 5 9BIRC5
Human telomerase reverse transcriptase

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

471 bp

F: CGACTACAGCCACTACTACACGA
R: CTCATCCGCAGAGCCTGT
F: CAGACACACGGTGAACTATGG
R: GATCAGCTTCCACTGTTAGACG
EGFR F: TCTCAGCCACATGTCGATGG
EGFR R: TCGCACTTCTTACACTTGCG
SURVIVIN F: TTAACCGCCAGATTTGAATCGCG
SURVIVIN R: GCTCCCAGCCTTCCAGCT
HTERTF: CGGAAGAGTGTCTGGAGCAA
HTERT R: GGATGAAGCGGAGTCTGGA
F: CATCACTGCCACCCAGAAGA
R: TCCACCACCCTGTTGCTGTA

Leukemia Virus (M-MLV) Reverse Transcriptase (Invitrogen,

Carlsbad, CA, USA). A mixture containing 2 g total RNA (blood
samples) or 1 g total RNA (urine samples), with 500 g oligo
dT18 primer (Fermentas St.Leon-Rot, Germany), 0.5 mM
deoxynucleotides (HT Biotechnology LTD., Cambridge, UK) and
DEPC-treated water up to 12 l, was heated at 65C for 5 min and
then chilled on ice for an additional 5 min. A second mixture
containing RT buffer (50 mM Tris-HCl/pH 8.3, 75 mM KCl, 3 mM
MgCl2), 10 mM dithiothreitol, 20 units RNase OUT Recombinant
Ribonuclease Inhibitor (Invitrogen), was added to the first mixture
and then incubated at 37C for 2 min. In the next step, 150 units of
M-MLV RT was added up to a final volume of 20 l, and the
incubation continued at 37C for 50 min and the reaction was
completed by an inactivation step at 70C for 15 min. The resulting
complementary DNA was then used for multiplex PCR reaction.
PCR conditions. All PCR reactions were performed using Qiagen
Multiplex PCR Kit (Qiagen, Hilden, Germany) in a final reaction
volume of 25 l. The reaction mixture contained 1X Qiagen Multiplex
PCR Mix (HotStarTaq DNA pol, Multiplex PCR Buffer with 6 mM
MgCl2/pH 8.7, dNTP mix), 0.125 M CK19 primers, 0.125 M CK20
primers, 0.4 M EGFR primers, 0.3 SURVIVIN primers, 0,3
hTERT primers and DEPC-treated water up to 23 l. Complementary
DNA (2 l) was added to the reaction volume. Cycling conditions
were 95C for 15 min (1 cycle), 94C for 30 s, 57C for 90 s, 72C for
60 s (36 cycles), and 72C for 10 min (1 cycle).
For GAPDH detection, the PCR reaction was performed using
Qiagen Taq PCR Kit (Qiagen) in a final reaction volume of 15 l. The
reaction mixture contained 1 Qiagen PCR Buffer (2.5 units Taq DNA
pol, PCR Buffer with 1.5 mM MgCl2, 200 M dNTPs mix), 0.66 M
GAPDH primers and DEPC-treated water up to 14 l. of
Complementary DNA (1 l) was added to the reaction volume.
Cycling conditions were the same as for multiplex PCR.
PCR products were analyzed by electrophoresis in a 2% agarose
gel (ethidium bromide stained) and were then captured under UV
light in KODAK EDAS 290 Imaging System (CareStream Health,
Rochester, NY, USA).
We proceed to a qualitative analysis. Before developing multiplex
PCR, first we perfomed single PCR for each gene using bladder
cancer tissue as a positive control and blood and urine from healthy
volunteers as negative control, normalizing the PCR so that there is

146 bp

no expression of any of these genes in the healthy controls. Blood

and urine samples with detectable GAPDH band together with the
positive marker product were considered positive for any gene
expression. Samples were considered negative when GAPDH-band
were identified alone. In cases of missing GAPDH signal , blood
and urine samples were excluded from analysis.
Statistical analysis. Quantitative variables are expressed as mean
valuesstandard deviation (SD)/median values (interquartile range).
Qualitative variables are expressed as absolute and relative
frequencies. For comparisons of proportions, chi-square and Fishers
exact tests were used. Analysis of variance (ANOVA) was used for
the comparison of mean age between the study groups.
KaplanMeier survival estimates for recurrence and disease
progression were graphed over the follow-up period for the study
groups. Recurrence-free survival was defined as the time from
diagnosis of bladder cancer to the date of the first bladder
recurrence (same or lower disease stage or grade). Progression-free
survival was defined as the time from diagnosis of bladder cancer to
the date that higher disease grade or stage were detected. Log-rank
tests were used for the comparison of survival curves. Sensitivity,
specificity, negative and positive predictive values were calculated to
estimate the discriminative ability of study markers between the
group with history of bladder cancer and controls (no cancer) as
well as between the newly diagnosed group and controls. All
reported p-values are two-tailed. Statistical significance was set at
p<0.05 and analyses were conducted using the Statistical Package
for the Social Sciences Predictive Analysis Software (SPSS PASW
statistics software version 19.0; IBM SPSS Statistics, IBM
Corporation,. Chicago, IL, USA).

Results
The proportion of men was 85.7%, 90.6% and 74.4% in the
newly-diagnosed group, in patients with history of bladder
cancer and in healthy volunteers, respectively (p=0.077).
Twenty-three patients (21.9%) from the newly-diagnosed
group underwent radical cystectomy and the corresponding
proportion was 12.5% for the group with history of bladder
cancer (p=0.125). None of the patients had distal metastasis,

7417

ANTICANCER RESEARCH 34: 7415-7424 (2014)

Table III. Expression of all study markers and their combinations in blood and urine in the three study groups.
Group

Blood
EGFR
CK19
CK20
EGFR and CK19
EGFR and CK20
CK19 and CK20
EGFR and CK19 and CK20
EGFR-CK19-positive
EGFR-CK20-positive
CK19-CK20-positive
EGFR-CK19-CK20-positive
Urine
SURVIV
hTERT
CK20
SURVIV and hTERT
SURVIV and CK20
hTERT and CK20
SURVIV and hTERT and CK20
SURVIV-hTERT-positive
SURVIV-CK20-positive
hTERT-CK20-positive
SURVIV-hTERT-CK20-positive

Newly-diagnosed
n (%)

History of bladder cancer

n (%)

Control
n (%)

p-Value

24
61
56
17
21
41
17
68
59
76
79

(23.1).C
(58.7)C
(53.8)C
(16.3)
(20.2).C
(39.4)
(16.3)
(65.4)C
(56.7)C
(73.1)C
(76.0)C

5
32
42
2
3
27
1
35
44
47
48

(8.2)
(50.0)
(65.6)C
(3.1)
(4.7)
(42.2)
(1.6)
(54.7)C
(68.8)C
(73.4)C
(75.0)C

2
12
12
2
2
8
2
12
12
17
17

(5.1)
(30.8)
(30.8)A,B
(5.1)
(5.1)
(20.5)
(5.1)
(30.8)A,B
(30.8)A,B
(43.6)A,B
(43.6)A,B

0.010*
0.028*
0.003*
0.027**
0.010**
0.090*
0.007**
0.003*
0.001*
0.004*
0.001*

22
11
35
9
22
9
9
25
35
38
38

(21)C
(10.5)
(33.3)C
(8.6)
(21)C
(8.6)
(8.6)
(23.8)C
(33.3)C
(36.2)C
(36.2)C

13
5
22
4
13
4
4
14
22
23
23

(20.3)C
(7.8)
(34.4)C
(6.3)
(20.3)C
(6.3)
(6.3)
(21.9)C
(34.4)C
(35.9)C
(35.9)C

2
0
1
0
1
0
0
2
2
1
2

(5.1),
(0.0)
(2.6),
(0)
(2.6),
(0)
(0)
(5.1),
(5.1),
(2.6),
(5.1),

0.043*
0.099**
<0.001*
0.175**
0.026*
0.175**
0.175**
0.038*
0.002*
<0.001*
0.001*

*Pearsons Chi-square test; **Fishers exact test. A,B,Cindicate significant differences in pairwise comparisons.

whereas positive nodes were found in 7.6% of the newlydiagnosed group and 3.1% of the group with history of
bladder cancer (p=0.322). Of the newly-diagnosed group,
17.2% were categorized as having disease of anatomic stage
III or IV (AJCC) and the corresponding proportion was 4.7%
for the group with history of bladder cancer (p=0.017).
Recurrence was recorded in 20 patients (19.2%) from the
newly-diagnosed group and in 26 patients (40.6%) from the
group with history of bladder cancer (p=0.002), while
disease progression was recorded in 12 of the patients with
newly-diagnosed bladder cancer (11.8%) and in 11 (18.0%)
with history of bladder cancer (p>0.050). The mean followup period was 26.9 months (SD=11.9), with a median of 32
months (interquartile range from 15 to 37 months).
Expression of EGFR, CK19, CK20 in peripheral blood. The
detection of all markers and their respective combinations in
blood and urine samples in the three study groups is
presented in Table III.
With regard to blood measurements, EGFR-positive
detections were more frequent in the group of patients with

7418

newly-diagnosed bladder cancer compared to the group with

a history of bladder cancer and to healthy volunteers. CK19
detections were more frequent in the newly-diagnosed group
compared to the control group, whereas CK20-positive
detections were less frequent in the control group compared
to the two patient groups. The combination of positive
detection for both EGFR and CK19 was more frequent in
the newly-diagnosed group compared to the group of
patients with history of bladder cancer, while the
combination of both EGFR and CK20 was more frequent in
newly diagnosed as compared to group with history with
bladder cancer and the control group. Triple-positive
detections for EGFR plus CK19 plus CK20 were more
frequent in the newly-diagnosed group compared to the
group of patients with a history of bladder cancer.
EGFRCK19-positive detections were rare in control group
compared with newly-diagnosed and patients with history
of bladder cancer. Furthermore, the combined positive
detections of EGFR and CK20, CK19 and CK20, and EGFR
with CK19 and CK20 were rare in the control group as
compared with patient groups.

Leotsakos et al: CTCs in Bladder Cancer Using Multiplex PCR

Expression of hTERT, CK20 and SURVIVIN in urine. Urine

detection of SURVIVIN, CK20, the combined positive
detections of SURVIVIN plus CK20, SURVIV-hTERTpositive, SURVIV-CK20-positive, hTERT-CK20-positive and
SURVIV-hTERT-CK20-positive were frequent in the newlydiagnosed group and the group of patients with history of
bladder cancer, and rare in the control group (Table III).
Sensitivity, specificity, negative and positive predictive value
of markers used for discriminating newly-diagnosed and
control groups. Most of the markers and their respective
combinations in blood revealed high specificity and
predictive values (Table IV). EGFR detection showed low
sensitivity, while the sensitivity of CK19 and CK20 detection
was 58.7% and 53.9%, respectively. The highest sensitivity
was found for the combined positive detections of EGFR and
CK19 (65.4%), CK19 and CK20 (73.1%), and EGFR-CK19CK20-positive (76%).
Urinary markers and their combinations had high positive
predictive values ranging from 91.7% to 100% and high
specificity rates ranging from 94.9% to 100%. The sensitivity
was low, the highest being detected for combined positive
detection of hTERT-CK20-positive (35.9%), and SURVIVINhTERT-CK20-positive (36.2%).
Sensitivity, specificity, negative and positive predictive values
of marker detections in patients with history of bladder
cancer and control group. Most of the markers and their
respective combinations in blood showed high sensitivity and
negative predictive values (Table V). The highest sensitivity
rates concerning blood measures were found for CK20
(65.6%), EGFR and CK20 (68.8%), CK19 and CK20
(73.4%), and EGFR-CK19-CK20-positive (75.0%).
Urinary markers also presented high specificity and
positive predictive values, while the highest sensitivity rates
were found for CK20, SURVIV-CK20-positive, hTERTCK20-positive and SURVIV-hTERT-CK20-positive.
Association with progression-free survival. Patients in the
newly-diagnosed bladder cancer group with positive urine
SURVIVIN detection had worse progression-free survival
compared to those that were negative for SURVIVIN (logrank test, p=0.011) (Figure 1). Specifically, three of the
newly-diagnosed patients with negative SURVIVIN (8.8%)
had disease progression and the corresponding proportion
was 26.9% in cases that SURVIVIN was detected. The
positive detection of urinary CK20 was also associated with
worse progression free-survival in newly diagnosed patients
(log-rank test, p=0.050) (Figure 2).
Worse progression free-survival was found in the newlydiagnosed group with combined positivity for the detection
of urinary SURVIV and hTERT (log-rank test, p=0.048),
urinary SURVIV and CK20 (log-rank test, p=0.011),

urinary hTERT and CK20 (log rank test, p=0.048), urinary

SURVIV with hTERT and CK20 (log-rank test, p=0.048),
urinary SURVIV and hTERT (log-rank test, p=0.029), and
urinary SURVIV and CK20 (log-rank test, p=0.050). In
addition, recurrence-free rates were worse in the cases of
newly diagnosed patients with combined positivity for urinary
SURVIVIN and hTERT (log-rank test, p=0.050), urinary
hTERT and CK20 (log-rank test, p=0.050), and urinary
SURVIVIN plus hTERT plus CK20 (log-rank test, p=0.048).
Correlation of positive detections with various clinicopathological parameters of patients with bladder cancer. In the
newly-diagnosed group, EGFR was more frequently expressed
in patients with histological grade 3 or carcinoma in situ
compared to those with histological grade <3 (31.4% vs.
15.1%, p=0.049). In this group, the positive detection of CK20
in blood was correlated with histological grade 3 or carcinoma
in situ as compared to those with histological grade <3 (68.6%
vs. 39.6%, p=0.003). Similarly, the combined positive detection
in blood of EGFR and CK20 (72.5% vs. 41.5%, p=0.001) and
EGFR-CK19-CK20-positive (84.3% vs. 67.9%, p=0.050)
correlated with histological grade 3 or carcinoma in situ.
Furthermore, in newly-diagnosed patients, positive hTERT
detection in urine was associated with primary tumor size 3
(that is T3 or T4) compared to those with tumor size <3 (that
is T1 or T2) (28.6% vs. 7.7%, p=0.032) and with AJCC stage
2 or greater, compared to stage <2 (50.0% vs. 6.3%,
p=0.028). Similarly, the combined positive detection of
SURVIVIN and hTERT (50.0% vs. 6.3%, p=0.028), hTERT
and CK20 (50.0% vs. 6.3%, p=0.028), and SURVIVIN with
hTERT and CK20 (50.0% vs. 6.3%, p=0.028) were
associated with AJCC stage 2 or greater.
In addition, all marker detections in urine samples and
their respective combinations were associated with
histological grade 3 or carcinoma in situ (for SURVIVIN
31.4% vs. 11.1%, p=0.011; for hTERT: 19.6% vs. 1.9%,
p=0.003; and for CK20: 54.9% vs. 13.0%, p<0.001).
In the blood samples of the group of patients with history
of bladder cancer, the detection of CK20 (80.0% vs. 52.9%,
p=0.023) and the combined positive detection of EGFR and
CK20 (83.3% vs. 55.9%, p=0.018), CK19 and CK20 (86.7%
vs. 61.8%, p=0.024), and EGFR with CK19 and CK20
(90.0% vs. 61.8%, p=0.009) correlated with histological
grade 3 or carcinoma in situ as compared to those with
histological grade <3, respectively.

Discussion
Although the European Organization for Research and
Treatment of Cancer (EORTC) Genito-Urinary Cancer
Group has developed a scoring system and risk tables (26),
more intensive research is required in the field of CTCs in
order to improve the predictive accuracy of the currently

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ANTICANCER RESEARCH 34: 7415-7424 (2014)

Table IV. Percentage sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of study markers for the discrimination between
newly-diagnosed patients with bladder cancer and controls.
Sensitivity (95% CI)
Blood
EGFR
CK19
CK20
EGFR and CK19
EGFR and CK20
CK19 and CK20
EGFR and CK19 and CK20
EGFR-CK19-positive
EGFR-positiveCK20-positive
CK19-CK20-positive
EGFR-CK19-CK20-positive
Urine
SURVIV
hTERT
CK20
SURVIV and hTERT
SURVIV and CK20
hTERT and CK20
SURVIV and hTERT and CK20
SURVIV-hTERT-positive
SURVIV-positiveCK20-positive
hTERT CK20-positive
SURVIV-hTERT-CK20-positive

Specificity (95% CI)

23.1
58.7
53.9
16.4
20.2
39.2
16.4
65.4
56.7
73.1
76.0

(15.4-32.4)
(48.6-68.2)
(43.8-63.7)
(9.8-24.9)
(13.0-29.2)
(30.0-49.5)
(9.8-24.9)
(55.4-74.5)
(46.7-66.4)
(63.5-81.3)
(66.6-83.8)

94.9
69.2
69.2
94.9
94.9
79.5
94.9
69.2
69.2
56.4
56.4

21.0
10.5
33.3
8.6
21.0
8.6
8.6
23.8
33.3
36.2
36.2

(13.6-30.0)
(5.4-18.0)
(24.4-43.2)
(4.0-15.7)
(13.6-30.0)
(4.0-15.7)
(4.0-15.7)
(16.0-33.1)
(24.4-43.2)
(27.0-46.2)
(27.0-46.2)

94.9
100.0
97.4
100.0
97.4
100.0
100.0
94.9
94.9
97.4
94.9

(82.7-99.4)
(52.4-83.0)
(52.4-83.0)
(82.7-99.4)
(82.7-99.4)
(63.5-90.7)
(82.7-99.4)
(52.4-83.0)
(52.4-83.0)
(39.6-72.2)
(39.6-72.2)
(82.7-99.4)
(91.0-100.0)
(86.5-99.9)
(91.0-100.0)
(86.5-99.9)
(90.97-100.0)
(90.97-100.0)
(82.7-99.4)
(82.7-99.4)
(86.5-99.9)
(82.7-99.4)

PPV (95% CI)

NPV (95% CI)

92.3
83.6
82.4
89.5
91.3
83.7
89.5
85.0
83.1
81.7
82.3

(74.9-99.1)
(73.1-91.2)
(71.2-90.5)
(66.9-98.7)
(72.0-98.9)
(70.3-92.7)
(66.9-98.7)
(75.3-92.0)
(72.3-91.0)
(72.4-89.0)
(73.2-89.3)

31.6
38.6
36.0
29.8
30.8
33.0
29.8
42.9
37.5
44.0
46.8

(23.3-40.9)
(27.2-51.0)
(25.2-47.9)
(22.0-38.7)
(22.7-39.9)
(23.6-43.4)
(22.0-38.7)
(30.5-56.0)
(26.4-49.7)
(30.0-58.8)
(32.1-61.9)

91.7
100.0
97.2
100.0
95.7
100.0
100.0
92.6
94.6
97.4
95.0

(73.0-99.0)
(71.5-100.0)
(85.5-99.9)
(66.4-100.0)
(78.1-99.9)
(66.4-100.0)
(66.4-100.0)
(75.7-99.1)
(81.8-99.3)
(86.5-99.9)
(83.1-99.4)

30.8
29.3
35.2
28.9
31.4
28.9
28.9
31.6
34.6
36.2
35.6

(22.7-39.9)
(21.8-37.8)
(26.2-45.0)
(21.4-37.3)
(23.3-40.5)
(21.4-37.3)
(21.4-37.3)
(23.3-40.9)
(25.7-44.4)
(27.0-46.2)
(26.4-45.6)

Table V. Percentage sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of study markers for the discrimination between the
group with history of bladder cancer and controls.
Sensitivity (95% CI)
Blood
EGFR
CK19
CK20
EGFR and CK19
EGFR and CK20
CK19 and CK20
EGFR and CK19 and CK20
EGFR-CK19-positive
EGFR-CK20-positive
CK19-CK20-positive
EGFR-CK19-CK20-positive
Urine
SURVIV
H-TERT
CK20
SURVIV and H-TERT
SURVIV and CK20
H-TERT and CK20
SURVIV and H-TERT and CK20
SURVIVN-H-TERT-positive
SURVIV-CK20-positive
H-TERT-CK20-positive
SURVIV-H-TERT-CK20-positive

7420

Specificity (95% CI)

PPV (95% CI)

NPV (95% CI)

8.2
50.0
65.6
3.1
4.7
42.2
1.6
54.7
68.8
73.4
75.0

(2.7-18.1)
(37.2-62.8)
(52.7-77.1)
(0.4-10.8)
(1.0-13.1)
(29.9-55.2)
(0.0-8.4)
(41.8-67.2)
(55.9-79.8)
(60.9-83.7)
(62.6-85.0)

94.9
69.2
69.2
94.9
94.9
79.5
94.9
69.2
69.2
56.4
56.4

(82.7-99.4)
(52.4-83.0)
(52.4-83.0)
(82.7-99.4)
(82.7-99.4)
(63.5-90.7)
(82.7-99.4)
(52.4-83.0)
(52.4-83.0)
(39.6-72.2)
(39.6-72.2)

71.4
72.7
77.8
50.0
60.0
77.1
33.3
74.5
78.6
73.4
73.9

(29.0-96.3)
(57.2-85.0)
(64.4-88.0)
(6.8-93.2)
(14.7-94.7)
(59.9-89.6)
(0.8-90.6)
(59.7-86.1)
(65.6-88.4)
(60.9-83.7)
(61.5-84.0)

39.8
45.8
55.1
37.4
37.8
45.6
37.0
48.2
57.5
56.4
57.9

(29.8-50.5)
(32.7-59.3)
(40.2-69.3)
(27.9-47.7)
(28.2-48.1)
(33.5-58.1)
(27.6-47.2)
(34.7-62.0)
(42.2-71.7)
(39.6-72.2)
(40.8-73.7)

20.3
7.8
34.4
6.3
20.3
6.3
6.3
21.9
34.4
35.9
35.9

(11.3-32.2)
(2.6-17.3)
(23.0-47.3)
(1.7-15.2)
(11.3-32.2)
(1.7-15.2)
(1.7-15.2)
(12.5-34.0)
(23.0-47.3)
(24.3-48.9)
(24.3-48.9)

94.9
100.0
97.4
100.0
97.4
100.0
100.0
94.9
94.9
97.4
94.9

(82.7-99.4)
(91.0-100.0)
(86.5-99.9)
(91.0-100.0)
(86.5-99.9)
(91.0-100.0)
(91.0-100.0)
(82.7-99.4)
(82.7-99.4)
(86.5-99.9)
(82.7-99.4)

86.7
100.0
95.7
100.0
92.9
100.0
100.0
87.5
91.7
95.8
92.0

(59.5-98.3)
(47.8-100.0)
(78.1-99.9)
(39.8-100.0)
(66.1-99.8)
(39.8-100.0)
(39.8-100.0)
(61.6-98.5)
(73.0-99.0)
(78.9-99.9)
(74.0-99.0)

42.1
39.8
47.5
39.4
42.7
39.4
39.4
42.5
46.8
48.1
47.5

(31.6-53.1)
(30.0-50.2)
(36.2-59.0)
(29.7-49.7)
(32.3-53.6)
(29.7-49.7)
(29.7-49.7)
(32.0-53.6)
(35.5-58.4)
(36.7-59.6)
(36.0 9.1)

Leotsakos et al: CTCs in Bladder Cancer Using Multiplex PCR

Figure 1. Kaplan-Meier estimates for disease progression in the newlydiagnosed group according to the expression of SURVIV.

Figure 2. KaplanMeier estimates for disease progression in the group

of patients with newly-diagnosed bladder cancer according to the
detection of CYTOKERATINE 20 (CK20).

existing risk tables (27, 28). The goal would be not only to
use these molecular markers as a screening test in
populations at higher risk for bladder cancer (29, 30), but to
also achieve a better surveillance (31-34) and perhaps a
better understanding over the biological behavior of the
disease, especially when there is interobserver variability in
classification of stage T1 versus Ta tumors and tumor
grading in both 1997 and 2004 classifications. In such
difficult cases, where an additional review by an experienced
genitourinary pathologist is recommended, an objective,
easy-to-perform and highly specific assay would be of great
importance.
Rink et al. showed significantly worse overall
progression-free survival and cancer-specific survival in
CTC-positive compared to CTC-negative patients (35).
Msaouel and Koutsilieris further performed a systematic
review and meta-analysis of the value of CTC detection in
bladder and urothelial cancer, highlighting the potential
clinical role of CTCs as markers of advanced bladder
cancer (17). It has also been shown that CTC status may
provide prognostic information in the face of negative
imaging (36). In the case of urothelial carcinoma, it could
provide clinicians with valuable information that might be
applied to preoperative management algorithms.
Furthermore, CTC status could potentially identify patients
who would be most appropriate for neoadjuvant
chemotherapy or clinical trial protocols.

The advantages of multi-marker use for CTC detection in

patients with colorectal cancer (CRC) have already been
described (15, 18, 37, 38). Many previous studies have
shown that the use of more than one marker independently
increases the sensitivity and specificity of CTC detection
and thus the correlation with disease stage in patients with
CRC (39-43). Implementation of multiplex RT-PCR, which
allows for examination of multiple marker expression in a
single reaction, could be advantageous, in contrast with the
application of separate PCR reactions for each marker,
which can be time-consuming and costly. Moreover, the use
of multiplex RT-PCR on peripheral blood and urine samples
represents an easily applied, non-invasive technique for the
detection of CTCs in patients with cancer (44).
The aim of the present study was to develop and then
evaluate the clinical significance of a multiplex PCR-based
detection of CTCs using primers for CK19, CK20 and EGFR
(in blood samples) and SURVIVIN, hTERT and CK20 (in
urine samples) and to assess clinical applications of these
markers in patients with bladder cancer. With a substantial
follow-up period, we were able to estimate several clinical
parameters (sensitivity, specificity, positive predictive value,
negative predictive value, progression-free survival) and
assess their prognostic relevance.
Our data highlight the known correlation between
primary tumor (T stage) and high grade (grade 3 or
carcinoma in situ) with worst prognosis regarding

7421

ANTICANCER RESEARCH 34: 7415-7424 (2014)

recurrence and progression in patients with bladder cancer.
Beyond these, there seems to be a statistically significant
correlation of those clinical parameters with the some of the
evaluated mRNA markers.
There is considerable heterogenicity in tumor markers
used in several studies. From the markers we used, EGFR
and CK20 appeared to be the most commonly used markers
in many studies. Positive EGFR detection in blood samples
demonstrated high specificity (and positive predictive values)
for diagnosis of bladder cancer, both in the newly-diagnosed
group and in patients with history of bladder cancer. CK20
had relatively the highest sensitivity rates in both groups,
both in blood and urine samples. These results seem to agree
with those from relevant studies (17). On the other hand,
EGFR demonstrated low diagnostic sensitivity rates
compared with all other markers tested. Urine markers
demonstrated also high specificity and positive predictive
values, both in the newly diagnosed group and in patients
with history of bladder cancer. Therefore CTCs detection
may be useful in confirming the cancer diagnosis.
Positive SURVIVIN detection in urine of newly-diagnosed
patients, revealed worse prognosis. The positive detection of
CK20 in urine samples was also associated with worse
progression free-survival in newly-diagnosed patients. In the
same group, EGFR, as well as CK2O detection in blood
samples (and their combination in group of patients with
history of bladder cancer) was more frequently expressed in
patients with high tumor grade (histological grade 3 or
carcinoma in situ). Furthermore, in newly-diagnosed,
positive hTERT detections correlated more with primary
tumor size (T) 3 and with AJCC stage 3 or greater as
compared with stage <3. In addition, all marker detections
in urine samples and their respective combined positivity
correlated with high-grade cancer (histological grade 3 or
carcinoma in situ), in the newly-diagnosed patients. There is
thus a considerable connection with the conclusion of Rink
et al., that that the presence of CTC may be predictive for
early systemic disease (35).
Multiplex RT-PCR assay can provide useful information
concerning bladder cancer stage, grading and progressionfree and recurrence-free survival. The combination of the
mRNA markers in a single reaction could be beneficial in
the early detection of high-grade/clinically relevant disease
and monitoring of bladder cancer patients (mainly those
with high possibility to progress or recurrence), as it has
previously been documented in other malignancies (38, 43,
45-49).
This study does have several limitations. The overall
number of patients needs to be greater, therefore, the results
must be interpreted with caution. The usage of CTCs seems
promising, but for the moment is mainly study-based and no
large trials have been performed yet; therefore, no clinically
usable urinary markers have been identified. Relatively larger-

7422

scale studies and longer follow-up surveys would prove the

clinical significance and prognostic importance of multiplex
PCR-based detection of CTCs using EGFR, CK19 and CK20
in blood, hTERT, CK20 and SURVIVIN in urine and mainly
their combinations in patients with bladder cancer. Despite
the relatively low number of patients, to our knowledge, it is
one of the largest studies to date in patients with bladder
cancer. There is also a need for large quantitative studies,
which will allow us to evaluate the optimal cut-off CTC count
for increased sensitivity and specificity.
Another minor limitation is probably that of the healthy
control group. They may have had occult bladder cancer
which could somewhat confound interpretation of the results.
In bladder cancer, is not easy to find volunteers for
cystoscopy. People without clinical or laboratory suspicion,
without a known history of malignancy or who are operated
on for another entirely irrelevant reason (e.g. inguinal
hernia), can probably be considered as control group. For a
small proportion (n=6) of the control group (viz incidents
audited even with transurethral resection), despite the clinical
suspicion (random ultrasound scan, mostly for abdominal
pain, which showed a possible bladder tumor), the final
diagnosis (even with cystoscopy and random biopsies) was
negative. Although not be considered exactly as healthy
volunteers at the beginning, they proved to be healthy.
The future of marker development seems bright and new
techniques are emerging. Beyond finding good markers, the
financial costeffectiveness will be an important issue and
that has not been sufficiently studied yet. Currently, no single
marker can guide us in surveillance and lower the frequency
of urethrocystoscopy. Whether use of a set of markers will
be the answer will have to be studied.

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Received July 28, 2014

Revised September 12, 2014
Accepted September 18, 2014