EUROPEAN UROLOGY 64 (2013) 465–471

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Bladder Cancer

Prospective Evaluation of a Molecular Marker Panel for Prediction

of Recurrence and Cancer-specific Survival After Radical Cystectomy

Yair Lotan a,*, Aditya Bagrodia a, Niccolo Passoni b, Varun Rachakonda a, Payal Kapur c,
Yull Arriaga d, Christian Bolenz e, Vitaly Margulis a, Ganesh V. Raj a,
Arthur I. Sagalowsky a, Shahrokh F. Shariat f,g
a b
Department of Urology, University of Texas Southwestern Medical Center Dallas, TX, USA; Department of Urology, University Vita-Salute San Raffaele,
Milan, Italy; c Department of Pathology, University of Texas Southwestern Medical Center Dallas, TX, USA; d Division of Medical Oncology, University of Texas
e
Southwestern Medical Center Dallas, TX, USA; Department of Urology, Mannheim Medical Center, University of Heidelberg, Mannheim, Germany;
f
Department of Urology, Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY, USA; g Division of Medical Oncology, Weill Cornell
Medical College, New York Presbyterian Hospital, New York, NY, USA

Article info Abstract

Article history: Background: Retrospective studies demonstrated that cell cycle–related and prolifera-
Accepted March 24, 2013 tion biomarkers add information to standard pathologic tumor features after radical
Published online ahead of cystectomy (RC). There are no prospective studies validating the clinical utility of
markers in bladder cancer.
print on April 3, 2013 Objective: To prospectively determine whether a panel of biomarkers could identify
patients with urothelial carcinoma of the bladder (UCB) who were likely to experience
Keywords: disease recurrence or mortality.
Design, setting, and participants: Between January 2007 and January 2012, every patient
Bladder cancer
with high-grade bladder cancer, including 216 patients treated with RC and lymphadenec-
Molecular markers tomy, underwent immunohistochemical staining for tumor protein p53 (Tp53); cyclin-
Cystectomy dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A); cyclin-dependent kinase inhibitor
1B (p27, Kip1); antigen identified by monoclonal antibody Ki-67 (MKI67); and cyclin E1.
Intervention: Every patient underwent RC and lymphadenectomy, and marker staining.
Outcome measurements and statistical analysis: Cox regression analyses tested the
ability of the number of altered biomarkers to predict recurrence or cancer-specific
mortality (CSM).
Results and limitations: Pathologic stage among the study population was pT0 (5%), pT1
(35%), pT2 (19%), pT3 (29%), and pT4 (13%); lymphovascular invasion (LVI) was seen in 34%.
The median number of removed lymph nodes was 23, and 60 patients had lymph node
involvement (LNI). Median follow-up was 20 mo. Expression of p53, p21, p27, cyclin E1,
and Ki-67 were altered in 54%, 26%, 46%, 15%, and 75% patients, respectively. In univariable
analyses, pT stage, LNI, LVI, perioperative chemotherapy (CTx), margin status, and number
of altered biomarkers predicted disease recurrence. In a multivariable model adjusting for
pathologic stage, margins, LNI, and adjuvant CTx, only LVI and number of altered bio-
markers were independent predictors of recurrence and CSM. The concordance index of a
baseline model predicting CSM (including pathologic stage, margins, LVI, LNI, and adjuvant
CTx) was 80% and improved to 83% with addition of the number of altered markers.
Conclusions: Molecular markers improve the prediction of recurrence and CSM after RC.
They may identify patients who might benefit from additional treatments and closer
surveillance after cystectomy.
# 2013 European Association of Urology. Published by Elsevier B.V. All rights reserved.

* Corresponding author. Department of Urology, The University of Texas Southwestern Medical

Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390, USA. Tel. +1 214 648 0389;
Fax: +1 214 648 8786.
E-mail address: Yair.lotan@utsouthwestern.edu (Y. Lotan).
0302-2838/$ – see back matter # 2013 European Association of Urology. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.eururo.2013.03.043
466 EUROPEAN UROLOGY 64 (2013) 465–471
1. Introduction
Urothelial carcinoma of the bladder (UCB) is the fifth
most common cancer worldwide, with an estimated
73 510 cases and 14 880 deaths in the United States for
2012 [1]. Up to 75% of UCB patients are diagnosed with
non–muscle-invasive (NMI) disease, most of which can be
managed by transurethral resection (TUR) and intrave-
sical therapy. However, 30% of patients with high-grade
NMI UCB will eventually experience disease progression
to muscle-invasive (MI) disease [2]. For these patients
and the 25% of patients who present with primary MI
UCB, radical cystectomy (RC) and bilateral lymphadenec-
tomy with or without perioperative chemotherapy (CTx)
represents the best chance for cure [3–5]. Unfortunately,
>25% of patients classified as having favorable pathologic
features such as pathologic stages pT1–pT3a without
nodal involvement (pN0) at the time of RC experience
disease recurrence within 5 yr of their surgery. These
patients eventually develop metastasis and succumb to
their disease [3–5].
This variability in outcomes among patients illustrates
the inherent biologic and clinical heterogeneity of UCB.
The American Joint Committee on Cancer (AJCC) TNM
staging system, which currently guides UCB management,
is limited in its ability to accurately predict an individuals’
risk of disease recurrence and/or UCB-specific mortality
[6,7]. Even the combination of clinical and pathologic
variables in mathematical algorithms such as nomograms
reaches discrimination for UCB recurrence of only 78%
[6,7].
Over the last decade, various investigators have shown
that alterations in candidate protein expression profiles
may help refine the understanding of the biologic and
clinical behavior of UCB, thereby improving the risk
stratification and clinical management of UCB patients
[8]. The development of emerging biomarkers might be
considered a highly regulated process that is conceptually
similar to therapeutic drug evaluation [9].
Therefore, we have undertaken a phased, systematic
evaluation and validation of a panel of established biomark-
ers (cyclin E1; tumor protein p53 [Tp53; formerly p53];
cyclin-dependent kinase inhibitor 1A [p21, Cip1] [CDKN1A;
formerly p21]; cyclin-dependent kinase inhibitor 1B (p27,
Kip1); and antigen identified by monoclonal antibody Ki-67
[MKI67; formerly Ki-67]) selected based on extensive clinical
data supporting their importance in UCB progression and
metastasis [10–12]. As part of this staged approach, we have
established the potential of this panel for UCB prognostica-
tion in retrospective, single-institution studies and in large,
multi-institutional studies [11,13–16].
In the next phase, we initiated a prospective protocol to
test the prognostic value of these biomarkers through
improved staging of patients with high-grade UCB under-
going RC. If the additive value of the biomarkers panel is
confirmed in this prospective study, we plan to integrate
this panel in the design of clinical trials of adjuvant
therapies.
2. Materials and methods
All procedures described in the present study were undertaken with the
approval and oversight of the Institutional Review Board for the
Protection of Human Subjects. All patients were treated at University of
Texas Southwestern Medical Center Dallas, TX, USA.
2.1. Patient population
A prospective study was designed to perform immunohistochemical
staging for cyclin E1, Tp53, CDKN1A, p27, and MKI67 on all high-grade
UCB specimens at our institution starting in January 2007. This study
focused on 216 consecutive patients who underwent RC with bilateral
lymphadenectomy for UCB with curative intent. Preoperative imaging
included chest radiographs and computed tomography scans of the
abdomen and pelvis. Marker staining was performed on the TUR
specimen prior to RC on 36 specimens and the remaining 180 patients
had staining of their RC specimens. Overall, 48 patients (22%) underwent
neoadjuvant CTx and 29 (13%) underwent adjuvant CTx based on high-
risk features. Neoadjuvant CTx consisted of gemcitabine and cisplatinum
in 34 patients (two cycles [n = 2], three cycles [n = 17], four cycles
[n = 13], five cycles [n = 2]); methotrexate, vinblastine, doxorubicin, and
cisplatin (MVAC) in nine patients (two cycles [n = 1], three cycles [n = 5],
four cycles [n = 3]); one patient received gemzar/carboplatin for four
cycles; one patient received ifosfamide and adriamycin for three cycles;
one patient received ifosfamide/cisplatin/paclitaxel for four cycles; one
patient received four cycles of gemcitabine and carboplatin; and one
received cisplatin-based CTx, but unknown other drugs for three cycles.
Adjuvant therapy consisted of gemcitabine and cisplatinum for three
cycles (n = 5), four cycles (n = 17), five cycles (n = 2), or six cycles (n = 1);
MVAC was given to two patients (one for three cycles and one for four
cycles); carboplatin and taxol were given to one patient for four cycles;
and one patient was given carboplatin for one cycle followed by gemzar
for four cycles. None of the patients received prior radiation therapy of
the pelvis.
All UCB specimens were examined and evaluated by pathologists
with expertise in genitourinary diseases according to previously
described protocols [13,17]. The UCB specimens were staged and graded
according to the AJCC 2002 TNM classification and the 1998 World
Health Organization/International Society of Urologic Pathology classi-
fication, respectively [18]. Comprehensive clinical and pathologic
parameters of each patient were entered into an institutional review
board-approved prospective database.
All patients were followed according to a previously described
standardized protocol [3]. Briefly, patients were evaluated postopera-
tively at least every 3–4 mo for the first year, semiannually for the second
year, and annually thereafter. Follow-up visit consisted on physical
examination and serum chemistry evaluation. Computed tomography
scanning of abdomen and pelvis was performed every 6 mo and chest
radiography was performed every 3–4 mo in first 2 yr, then semiannually
or when clinically indicated.
2.2. Immunohistochemistry and scoring
Immunohistochemical stainings on serial sections from the same
paraffin-embedded tumor block were performed for cyclin E1, Tp53,
CDKN1A, p27, and MKI67 using the Dako Autostainer (Dako North
America Inc, Carpinteria, CA, USA). The staining and scoring protocols for
all antibodies were previously described in detail [13,15]. Specimens of
normal human urothelium from patients undergoing cystectomy for
nonmalignant disease served as internal controls. For semiquantitative
scoring, bright-field microscopy imaging coupled with an advanced
color detection software (Automated Cellular Imaging System, Clarient,
 EUROPEAN UROLOGY 64 (2013) 465–471 467

CA, USA) was used. Staining intensity and percentage of positive nuclei Table 1 – Clinical and pathologic characteristics of patients
per area measurements were evaluated using 10 random hotspots within
each specimen. Nuclear Tp53 immunoreactivity was considered altered n = 216
when samples demonstrated at least 10% nuclear reactivity. CDKN1A Age at surgery, yr, median (IQR) 70 (62–76)
immunoreactivity was considered altered when samples demonstrated Sex, no. (%)
<10% staining. Nuclear p27 and cyclin E were considered altered when Male 171 (79)
samples demonstrated <30% nuclear reactivity. MKI67 immunoreactivity Female 45 (21)
was considered altered when samples showed >10% nuclear reactivity. Time from TUR to RC, mo, median (IQR) 2.1 (1.3–4.3)
Clinical stage, no. (%)
Staining was done on tissue from the RC or transurethral resection of
cTa/Tis/T1 96 (44)
bladder tumor (TURBT) performed within 3 mo prior to cystectomy.
cT2 85 (39)
Based on the number of altered biomarkers, we used an established cT3/T4 19 (9)
prognostic score (PS) as previously reported [17]. An altered expression Unknown 16 (7)
of none to two biomarkers was considered favorable, whereas an Clinical grade, no. (%)
alteration of at least three biomarkers represented an unfavorable PS. Low 4 (2)
High 201 (93)
Unknown 11 (5)
2.3. Statistical analyses
Concomitant CIS at TUR, no. (%)
No 160 (74)
The Fisher exact test and the Mann-Whitney U test were used to Yes 42 (19)
evaluate associations between biomarker expression and clinicopath- Unknown 14 (6)
ologic parameters. The Kaplan-Meier method was used to explore Pathological stage, no. (%)
pT0 8 (4)
recurrence-free survival (RFS) and cancer-specific survival (CSS), and
pT1 78 (36)
group differences were assessed using the log-rank test. Univariable
pT2 43 (20)
and multivariable Cox regression analyses tested the association pT3 59 (27)
between different prognostic variables and RFS and CSS. A multivari- pT4 28 (13)
able, Cox regression model predicting either RFS or CSS was constructed Pathologic grade, no. (%)
accounting for pathologic stage (pT0 vs pT1 vs pT2 vs pT3–4), soft- 0 8 (4)
Low 2 (1)
tissue margin status, lymphovascular invasion (LVI), lymph node
High 206 (95)
invasion (LNI), adjuvant CTx, and number of altered biomarkers.
Concomitant CIS at final pathology, no. (%)
Furthermore, the change in discrimination resulting from the addition No 120 (56)
to a baseline model (including pathologic stage, margin status, LVI, LNI, Yes 96 (44)
and adjuvant CTx) of the status of each individual biomarker, or the Lymphovascular invasion, no. (%)
number of altered biomarkers, or the prognostic score was quantified No 143 (66)
using the Harell Concordance index [19]. All statistical tests were Yes 73 (34)
Lymph node metastases, no. (%)
performed with the use of Stata v.12 (Stata Corp, College Station, TX,
No 161 (75)
USA). All tests were two-sided with a significance level at 0.05.
Yes 55 (25)
Total removed lymph nodes, no., median (IQR) 23.0 (16.0–32.0)
3. Results Positive lymph nodes, no., median (IQR) 3.0 (1.0–10.0)
Lymph node density, median (IQR) 14.3 (7.7–42.1)
Soft-tissue margin status, no. (%)
3.1. Demographics Negative 200 (93)
Positive 16 (7)
The patient demographics are shown in Table 1. The median Neoadjuvant chemotherapy, no. (%)
No 168 (78)
patient age was 70 yr with most being men (n = 192, 79%).
Yes 48 (22)
Most patients’ cancers were in clinical stage cTa/Tis/T1 Adjuvant chemotherapy, no. (%)
(n = 96, 44%) and cT2 (n = 85, 39%). After RC, there was a No 187 (87)
significant upstaging, with 27% at stage pT3 (n = 59) and 13% Yes 29 (13)

at pT4 (n = 28) when compared to clinical staging at time of
TURBT. Concomitant carcinoma in situ was seen in 96 (44%)
patients and LVI in 73 (34%). The median number of removed
lymph nodes was 23 (interquartile range [IQR]: 16–32) and
LNI was seen in 55 patients (25%). Recurrence was noted in
64 patients after cystectomy with a median follow-up of
20 mo (IQR: 10–37 mo). Of the recurrences, 8 were local,
11 were local and distant, and 45 were distant only. RFS at 6,
12, and 24 mo was 85%, 76%, and 68%, respectively. Overall,
51 patients died of bladder cancer-related causes. CSS rates at
6, 12, and 24 mo was 95%, 84%, and 73%, respectively.
3.2. Marker status and univariable analysis
Marker expression and association with disease recurrence
is shown in Tables 2 and 3. Expression of Tp53, CDKN1A,
p27, cyclin E1, and MKI67 were altered in 54%, 26%, 46%,
IQR = interquartile range; TUR = transurethral resection; RC = radical
cystectomy; CIS = carcinoma in situ.
15%, and 75% patients, respectively. Each marker including
the number of altered markers, with the exception of
CDKN1A, was associated with recurrence after RC. The
numbers of altered markers, as well as individual markers
p27, Tp53, and MKI67, were associated with cancer-specific
mortality (CSM) after RC. Figures 1 and 2 show estimates
of disease recurrence-free and cancer-free survival proba-
bilities based on the number of altered markers demon-
strating worse outcomes with increasing number of altered
markers.
In univariable analyses for disease recurrence (Table 3),
pT3/T4, positive soft-tissue margins, LNI, LVI, perioperative
CTx, and number of altered biomarkers were predictors of
468 EUROPEAN UROLOGY 64 (2013) 465–471

Table 2 – Alterations of cell-cycle makers at radical cystectomy in the overall population and according to recurrence

Overall (n = 216) No recurrence (n = 152) Recurrence (n = 64) p value

Altered biomarkers, no. (%)

0 8 (4) 8 (5) 0 (0) 0.001
1 45 (21) 41 (27) 4 (6)
2 83 (38) 56 (37) 27 (42)
3 63 (29) 39 (26) 24 (38)
4 17 (8) 8 (5) 9 (14)
Altered biomarkers, no. (%)
2 136 (63) 105 (69) 31 (48) 0.004
>2 80 (37) 47 (31) 33 (52)
Cyclin E, no. (%)
Negative 183 (85) 123 (81) 60 (94) 0.017
Positive 33 (15) 29 (19) 4 (6)
CDKN1A, no. (%)
Negative 160 (74) 116 (76) 44 (69) 0.2
Positive 56 (26) 36 (24) 20 (31)
p27, no. (%)
Negative 117 (54) 89 (59) 28 (44) 0.046
Positive 99 (46) 63 (41) 36 (56)
Tp53, no. (%)
Negative 99 (46) 78 (51) 21 (33) 0.013
Positive 117 (54) 74 (49) 43 (67)
MKI67, no. (%)
Negative 54 (25) 53 (35) 1 (2) <0.0001
Positive 162 (75) 99 (65) 63 (98)

CDKN1A = cyclin-dependent kinase inhibitor 1A (p21, Cip1); p27 = cyclin-dependent kinase inhibitor 1B (p27, Kip1); Tp53 = tumor protein p53; MKI67 = antigen
identified by monoclonal antibody Ki-67.

Table 3 – Univariate analysis for recurrence and cancer-specific mortality

Recurrence Cancer-specific mortality

HR IQR p value HR IQR p value

Age 1 0.98–1.03 0.9 1 0.97–1.02 0.8

Sex 1.18 0.66–2.11 0.6 1.51 0.83–2.76 0.2
pT0 0.83 0.20–3.38 0.8 0.91 0.22–3.76 0.9
pT1 0.18 0.09–0.39 <0.0001 0.12 0.04–0.33 <0.0001
pT2 0.44 0.20–0.98 0.043 0.5 0.21–1.16 0.11
pT3–4 6.4 3.65–11.21 <0.0001 6.77 3.54–12.96 <0.0001
Soft-tissue margins 2.53 1.15–5.57 0.022 2.97 1.33–6.65 0.008
Concomitant CIS 1.25 0.76–2.05 0.4 1.46 0.84–2.54 0.2
LVI 5.74 3.40–9.68 <0.0001 6.77 3.70–12.42 <0.0001
LNI 4.58 2.78–7.53 <0.0001 5.38 3.09–9.40 <0.0001
Positive nodes, no. 1.04 1.00–1.07 0.031 1.03 1.00–1.07 0.07
LND 1.02 1.01–1.03 0.001 1.02 1.00–1.03 0.021
Neoadjuvant chemotherapy 1.88 1.09–3.23 0.023 1.64 0.88–3.05 0.12
Adjuvant chemotherapy 2.98 1.74–5.10 <0.0001 3.73 2.10–6.63 <0.0001
Bladder prognosis score 2.41 1.47–3.97 0.001 2.64 1.51–4.59 0.001
Altered biomarkers, no. 1.95 1.49–2.54 <0.0001 1.98 1.48–2.65 <0.0001
Cyclin E 0.35 0.13–0.97 0.044 0.33 0.10–1.05 0.061
CDKN1A 1.57 0.92–2.67 0.1 1.54 0.85–2.79 0.2
p27 1.88 1.14–3.11 0.013 1.99 1.14–3.49 0.016
Tp53 2.16 1.27–3.69 0.005 2.42 1.32–4.42 0.004
MKI67 29.08 4.02–210.29 0.001 2.86  1015 0.00* <0.0001

HR = hazard ratio; IQR = interquartile range; CIS = carcinoma in situ; LVI = lymphovascular invasion; LNI = lymph node invasion; LND = lymph node dissection;
CDKN1A = cyclin-dependent kinase inhibitor 1A (p21, Cip1); p27 = cyclin-dependent kinase inhibitor 1B (p27, Kip1); Tp53 = tumor protein p53; MKI67 = antigen
identified by monoclonal antibody Ki-67.
*
All patients who died of bladder cancer had altered MKI67.

disease recurrence after RC. These same factors were
predictors of CSM with the exception of neoadjuvant CTx use.
3.3. Multivariable analyses
In a multivariable model of disease recurrence after RC, only
LVI (hazard ratio [HR]: 2.37; 95% confidence interval [CI],
1.2–4.67; p = 0.013) and the number of altered biomarkers
(HR: 1.83; 95% CI, 1.34–2.52; p = 0.0002) were independent
predictors of disease recurrence, after adjusting for the
effects of for pathologic stage, margin status, LNI, and
adjuvant CTx (Table 4). Only LVI (HR: 2.8; 95% CI, 1.29–6.05;
p = 0.009) and the number of altered markers (HR: 2.05;
95% CI, 1.42–2.96; p = 0.0001) were independent predictors
 EUROPEAN UROLOGY 64 (2013) 465–471 469

Table 4 – Multivariate analysis for recurrence and cancer-specific mortality

Recurrence Cancer-specific mortality

HR 95% CI p value HR 95% CI p value

Pathologic Stage
pT0 Ref – – Ref – –
pT1 0.25 0.05–1.22 0.088 0.17 0.03–0.96 0.044
pT2 0.24 0.05–1.23 0.087 0.23 0.04–1.23 0.086
pT3-T4 1.02 0.23–4.60 1 0.93 0.21–4.11 0.9
Soft-tissue margins 0.57 0.25–1.31 0.2 0.63 0.27–1.46 0.3
LVI 2.37 1.20–4.67 0.013 2.8 1.29–6.05 0.009
LNI 1.51 0.77–2.95 0.2 1.34 0.64–2.81 0.4
Adjuvant chemotherapy 0.79 0.41–1.54 0.5 0.99 0.50–1.99 1
Altered biomarkers. no. 1.83 1.34–2.52 0.0002 2.05 1.42–2.96 0.0001

HR = hazard ratio; CI = confidence interval; Ref = reference; LVI = lymphovascular invasion; LNI = lymph node invasion.

of CSM (all p < 0.01) after adjusting for the effects of
pathologic stage, margin status, LNI, and adjuvant CTx. A
separate multivariable analysis adjusting for pathologic
stage, margin status, LNI, and neoadjuvant CTx also found
that LVI and number of altered markers were independent
predictors of recurrence and CSM, while neoadjuvant CTx
[(Fig._1)TD$IG]
was not an independent predictor of recurrence or CSM.
The concordance index of a baseline model predicting
disease recurrence (including pathologic stage [pT0 vs pT1
vs pT2 vs pT3/T4], soft-tissue margins, LVI, LNI, and
adjuvant CTx) was 79%. The addition of the number of
altered markers improved prediction by 3% to 82%. The
addition of MKI67 alone improved prediction of disease
recurrence to 84%, which was more than any other marker
alone, since only one patient with MKI67 negative status
experienced disease recurrence. The concordance index
of the baseline model predicting CSM was 80%. The addition
of the number of altered markers improved prediction by
3% to 83%. The addition of MKI67 alone improved prediction
of CSM to 85%, which was more than any other marker
alone, since every patient who died of UCB had altered
MKI67 status. The addition of Tp53 status alone only added
1% to the prediction of disease recurrence and CSM.

4. Discussion

The utility and importance of biomarkers has been recog-

Fig. 1 – Kaplan-Meier curve depicting recurrence-free survival according
to number of altered biomarkers ( p = 0.004). alt. = altered;
BM = biomarker.
[(Fig._2)TD$IG]
Fig. 2 – Kaplan-Meier curve depicting cancer-specific survival according
to number of altered biomarkers ( p < 0.0001). alt. = altered;
BM = biomarker.
nized and biomarker discovery efforts are now common in
both academic and industrial settings. Yet despite intensified
interest and investment, few novel biomarkers are used in
clinical practice [9]. The reasons for this disjunction are
manifold and reflect the long and difficult pathway from
candidate biomarker discovery to clinical assay, and the lack
of coherent and comprehensive processes for biomarker
development. The development of various emerging bio-
markers might be considered a highly regulated process that
is conceptually similar to therapeutic drug evaluation,
especially from the point where it enters human testing.
We have adhered to a formal structure and phased
approach to guide the process of biomarker development
and validation [9]. First, we performed single-center,
retrospective, hypothesis-generating, human studies test-
ing the value of the biomarker panel shown in this article
[10–12]. Subsequently, our group found that a marker
panel performed better than individual markers and
improved prediction of disease recurrence and mortality
when modeled together with conventional clinicopatho-
logic features [17]. In the latter study, we found that a
panel of cyclin E1, Tp53, CDKN1A, p27, retinoblastoma
protein (pRb), and MKI67 provided discriminatory infor-
mation that significantly improved the ability to predict
470 EUROPEAN UROLOGY 64 (2013) 465–471
recurrence and UCB-specific survival in 191 pT1–pT3 pN0
patients who underwent RC [17]. We recently externally
validated the value of this panel in large, retrospective,
multicenter studies [15–20]. The current study represents
the culmination of these efforts into a prospective clinical
trial of patients with high-grade UCB. There are scant
prospective data available in oncology, and the clinical
utility of few biomarkers is established [21–25].
We found that expression of a higher number of altered
biomarkers was an independent predictor of disease
recurrence and CSM after RC along with LVI, after adjusting
for the effects of pathologic stage, margin status, LNI, and
adjuvant CTx. Furthermore, the concordance index of a
baseline model including pathologic stage, margin status,
LVI, LNI, and adjuvant CTx improved from 79% to 82% after
adding the number of altered biomarkers. These findings
support prior retrospective studies showing that assess-
ment of biomarker status may identify patients at high risk
for cancer recurrence who may benefit from perioperative
CTx and closer surveillance. We further confirmed the
importance of MKI67 in identifying patients at high risk for
CSM [13,14]. Every patient in our cohort who died of their
disease had altered MKI67. However, since a large propor-
tion of patients (65%) without recurrence also had altered
MKI67, MKI67 positivity alone has limited utility. In
contrast, MKI67 negativity may have significant utility.
Prospective studies evaluating the utility of molecular
biomarkers in surgically treated UCB are still sparse. Two
studies in NMI UCB evaluated the utility of Tp53 protein
overexpression and Tp53 gene mutation in predicting
recurrence and progression and showed no predictive value
[22,23]. Another large study in NMI UCB reported that
mutations in fibroblast growth factor receptor 3 (FGFR3)
were associated with increased rates of disease recurrence,
but not disease progression or survival, in patients with
pTa UCB [21]. Regarding patients with advanced disease,
one companion study to the Southwestern Oncology Group
8710 trial (neoadjuvant CTx plus RC vs RC alone) evaluated
the prognostic role of MKI67, Tp53, and angiogenic changes
[25]. Tissue specimens were obtained before neoadjuvant
CTx from 42 patients randomized to the combination-
treatment arm and 52 randomized to RC alone. Patients
whose tumors had increased MKI67 expression had better
progression-free survival (median: 66 vs 12 mo; p = 0.063)
and a nonsignificant increase in overall survival (median:
73 mo vs 38 mo; p = 0.25). Tp53 expression and microvessel
density were not associated with outcomes. Unfortunately,
this study did not include the entire trial population,
and the small number of patients precluded further
subanalyses.
A multicenter, adjuvant CTx study randomized patients
with pT1/T2N0M0 disease whose tumors demonstrated
10% nuclear reactivity on centrally performed immuno-
histochemistry for Tp53 to three cycles of adjuvant MVAC
versus observation; Tp53-negative patients were observed
[24]. This study found no difference in recurrence based on
Tp53 status. These results are not surprising considering our
findings, which demonstrated that patients with only one
marker abnormality had favorable outcomes and that only
by knowing the number of altered markers could there be
more differentiation of an individual patient’s outcomes.
We and others have shown that investigating multiple
pathways, including downstream effectors (eg, of Tp53),
can enhance the predictive power provided by a single
marker [11,12,26]. Furthermore, Tp53 alone only added 1%
to the predictive accuracy of a model using stage, LVI, and
LNI.
Our prospective study has some limitations. There are
issues related to the reliability of immunohistochemical
biomarker assessment. They are highly dependent on a
range of poorly controlled variables, including choice of
antibody, antibody dilution, fixation techniques, and
inconsistency in specimen handling. However, we reduced
the number of variables in the immunohistochemical
techniques by using highly standardized protocols, includ-
ing established staining protocols and automated scoring
systems based on bright-field microscopy imaging coupled
with advanced color-detection software. These techniques
have been shown to improve experimental standardization
and staining reproducibility. Furthermore, the prospective
assessment of each specimen in a consecutive manner
eliminates any biases. One consideration is that not every
marker was equally useful. Cyclin E was only abnormal in a
small number of patients and CDKN1A was not an
independent predictor of recurrence or survival. The goal
of the trial was not to optimize the most useful set of
markers but to validate a previously developed panel and
support the principle that a higher cumulative number of
alterations predicts worse outcomes after cystectomy. It is
likely that other markers will perform equally well or
better. Finally, this was not a homogenous population;
rather, it reflected a real-world scenario with some patients
receiving perioperative CTx while others did not. This could
affect the performance of the markers in unpredictable
ways that may, in part, enhance or diminish their
significance.
5. Conclusions
Cell cycle–related and proliferation-related markers at time
of cystectomy improve the prediction of recurrence and
CSM after RC. Such a marker panel may help identify
patients who might benefit from additional treatments and
closer surveillance after cystectomy.
Author contributions: Yair Lotan had full access to all the data in the
study and takes responsibility for the integrity of the data and the
accuracy of the data analysis.
Study concept and design: Lotan, Shariat.
Acquisition of data: Lotan, Bagrodia, Rachakonda, Kapur, Arriaga, Bolenz,
Margulis, Raj, Sagalowsky, Shariat.
Analysis and interpretation of data: Lotan, Passoni, Kapur, Shariat.
Drafting of the manuscript: Lotan, Shariat.
Critical revision of the manuscript for important intellectual content: Lotan,
Bagrodia, Passoni, Rachakonda, Kapur, Arriaga, Bolenz, Margulis, Raj,
Sagalowsky, Shariat.
Statistical analysis: Lotan, Passoni, Shariat.
Obtaining funding: Lotan.
 EUROPEAN UROLOGY 64 (2013) 465–471
Administrative, technical, or material support: None.
Supervision: Lotan, Margulis, Raj, Sagalowsky, Shariat.
Other (specify): None.
Financial disclosures: Yair Lotan certifies that all conflicts of interest,
including specific financial interests and relationships and affiliations
relevant to the subject matter or materials discussed in the manuscript
(eg, employment/affiliation, grants or funding, consultancies, honoraria,
stock ownership or options, expert testimony, royalties, or patents filed,
received, or pending), are the following: None.
Funding/Support and role of the sponsor: The Simmons Cancer Center
provided funding for this study and was involved in data collection.
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