EURURO Vol. 72 No. 3

462 476

1. Introduction
Bladder cancer is a commonmalignancy, with 429 000 new
cases and 165 000 deaths annually . Approximately 75%
of patients are initially diagnosed with non–muscle-
invasive bladder cancer (NMIBC) . For NMIBC the 5-yr
recurrence rate is high (50–70%) and progression to
muscle-invasive bladder cancer (MIBC) is observed in
10–15% of cases Treatment of patients with NMIBC
includes a surveillance regimen of varying intensity for at
least 5 yr, depending on risk stratification for recurrence
and progression . The need for frequent and long-term
surveillance makes bladder cancer the most expensive
cancer to treat Clinical and histopathological risk
factors for disease progression to MIBC include stage,
grade, size, concomitant carcinoma in situ (CIS), tumour
multiplicity, and recurrence rate Risk factors are
incorporated into European Organisation for Research
and Treatment of Cancer (EORTC) risk tables , which
are widely used in the clinic. Patients with intermediate-
and high-risk NMIBC are treated with adjuvant intravesical
instillations of bacillus Calmette-Gue´ rin (BCG) or mitomy-
cin C for 1–3 yr . Adjuvant instillations reduce the risk of
recurrence and delay or prevent progression to MIBC
. However, tumours with similar histopathological
characteristics can have widely different molecular fea-
tures and may belong to distinct molecular subgroups with
different disease aggressiveness. Such subgroups, repre-
senting different clinical risks, have been identified in
NMIBC by us and others . Consequently, clinically
useful molecular tests for stratifying patients to treatment
and follow-up regimens beyond well-established clinical
risk factors are greatly needed. We previously validated a
microarray-based gene expression signature for disease
progression in an independent cohort of 294 patients with
NMIBC This signature was transferred to a 12-gene
real-time qualitative polymerase chain reaction (RT-qPCR)
assay for calculating a progression score Our aim here
was to validate the assay in a multicentre prospective
study.
2. Patients and methods
2.1. Patients, follow-up, and biological material
All patients gave their written informed consent, and the study was
approved by the scientific ethics committee in each country (for details
see ). The inclusion criteria were: (1) patients diagnosed with
NMIBC (primary and recurrent); and (2) patients not previously
diagnosed with MIBC. Patients were included from year 2008 to
2012 and followed according to national guidelines. Follow-up was
registered online from each clinical centre and censored at the time of
the most recent cystoscopy. Follow-up included evaluation of
progression to MIBC. Progression to MIBC and/or metastatic disease
was verified by pathological examination and measured from the time
of surgery for the tumour analysed to the time of the event. Patients
were censored for cystectomy with no verified progression to MIBC,
death, or discontinuation of follow-up. Representative diagnostic
tumour sections were re-evaluated by one expert uropathologist
(F.A.) using the American Joint Committee on Cancer recommendations
from 2002 and graded according to the WHO 2004 guidelines.
Pathology review was performed for 89% (760/851) of tumours, with
a concordance rate of 78% when staging was possible based on the
single reviewed tumour section (508/654). When only the original
grade (1973 system) was available (n = 43) this information was
translated into theWHO 2004 grading system (G1 = low, G3 = high, and
G2 [n = 17] were included as unknown grade). The highest stage
(original or reviewed) and re-evaluated grade information were used in
the study where possible. Tissue material from primary and recurrent
tumours was collected fresh from resection in each clinical centre,
embedded in Tissue-Tek O.C.T. and snap frozen in liquid nitrogen before
storage at 80 8C. In all centres, standardised procedures for sampling,
freezing, and shipment of samples were applied. All subsequent
analytical procedures were performed at the Department of Molecular
Medicine, Aarhus University Hospital.
2.2. RNA extraction and quality assessment
Two sections stained with haematoxylin and eosin (top and bottom)
were evaluated for the presence of carcinoma cells for each tumour. Only
tumours with a carcinoma cell percentage > 10% (average for the two
sections) were used. Total RNA was extracted from serial cryosections
using an RNeasy Mini Kit (Qiagen); no trimming or microdissection was
performed. All samples were quantified using an Infinite 200 PRO
Results and limitations: The progression score was significantly ( p [10_TD$DIFF]< 0.001) associatedwith
age, stage, grade, carcinoma in situ, bacillus Calmette-Gue´ rin treatment, European Organi-
sation for Research and Treatment of Cancer risk score, and disease progression. Univariate
Cox regression analysis showed that patients molecularly classified as high risk experienced
more frequent disease progression (hazard ratio 5.08, 95% confidence interval 2.2–11.6;
p [10_TD$DIFF]< 0.001). Multivariable Cox regression models showed that the progression score added
independent prognostic information beyond clinical and histopathological risk factors
(p [10_TD$DIFF]< 0.001), with an increase in concordance statistic from 0.82 to 0.86. The progression
score showed high correlation (R2
[9_TD$DIFF] = 0.85) between paired fresh-frozen and formalin-fixed
paraffin-embedded tumour specimens, supporting translation potential in the standard
clinical setting. A limitation was the relatively low progression rate (5%, 37/750 patients).
Conclusions: The 12-gene progression score had independent prognostic power beyond
clinical and histopathological risk factors, and may help in stratifying NMIBC patients to
optimise treatment and follow-up regimens.
Patient summary: Clinical use of a 12-gene molecular test for disease aggressiveness may
help in stratifying patients with non–muscle-invasive bladder cancer to optimal treatment
regimens.
# 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.
Progression risk
Prospective study
Real-time qualitative
polymerase chain reaction
Validation
E U R O P E A N U R O L O G Y 7 2 ( 2 0 1 7 ) 4 6 1 – 4 6 9 462