european urology 50 (2006) 1347–1356
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From Lab to Clinic
Partially Degraded RNA from Bladder Washing is a Suitable
Sample for Studying Gene Expression Profiles in Bladder
Cancer
Lourdes Mengual a, Moise`s Burset a, Elisabet Ars a, Marı´a Jose´ Ribal b, Juan Jose´ Lozano c,
Bele´n Minana c, Lauro Sumoy c, Antonio Alcaraz b,*
a
Molecular Biology Laboratory, Fundacio´ Puigvert, Universitat Auto`noma de Barcelona, Spain
Department of Urology, Hospital Clı´nic, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS),
Universitat de Barcelona, Spain
c
Microarray Laboratory, Program in Bioinformatics and Genomics, Centre de Regulacio´ Geno`mica, Barcelona, Spain
b
Article info
Abstract
Article history:
Accepted May 29, 2006
Published online ahead of
print on June 15, 2006
Objectives: To determine the impact of different levels of RNA degradation on gene
expression measurements and to ascertain if the gene expression profile obtained
from bladder washing (BW) correlates to that obtained from the related bladder
tumour (BT).
Methods: BT and BW RNAs from the same patient were heat shocked to obtain three
RNA degradation states, which were compared with intact RNAs from healthy
bladders by using complementary DNA (cDNA) microarrays. All samples were
amplified by means of a T3N9-based transcription method. In addition, four of
the differentially expressed genes in microarrays related to bladder cancer (KRT20,
IGF2, GSN, and CCL2) were analyzed in 36 tumoural and 14 control BW samples by
quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).
Results: A high percentage of overlapping differentially expressed genes were
detected between BT arrays (85–91%) and between BW arrays (78–93%). Furthermore,
the similarity between BW and BT arrays was relatively high and independent of the
RNA degradation state (52–60%). Finally, expression differences for the four selected
genes were confirmed in the vast majority of extended BW samples tested by qRT-PCR.
Conclusions: Our results showed that partially degraded RNA samples analyzed by
cDNA microarrays yielded gene expression profiles comparable to those obtained
using intact RNA. Moreover, BW RNA exhibited gene expression patterns similar to
those identified in the BT, indicating that BW is an appropriate sample for studying
gene expression profiles of BT using cDNA microarrays. In addition, qRT-PCR results
further support the suitability of BW for gene expression profiling and its potential
use for routine diagnostics.
# 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.
Keywords:
Bladder neoplasms
Gene expression profiling
Microarray analysis
Molecular diagnostic
techniques
Reverse transcriptase
polymerase chain reaction
* Corresponding author. Hospital Clı´nic, Villarroel, 170, 08036 Barcelona, Spain.
Tel. + 34 93 2275545; Fax: +34 93 2275545.
E-mail address: [email protected] (A. Alcaraz).
0302-2838/$ – see back matter # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2006.05.039
1348
1.
european urology 50 (2006) 1347–1356
Introduction
Optimal diagnosis of bladder cancer currently relies
on cystoscopy, which is an invasive method. Urinary
cytology supplements but cannot replace endoscopic evaluation [1].
As a noninvasive alternative for diagnosing bladder cancer, gene expression levels of several molecular tumour markers have been recently studied in
voided urine or bladder washing (BW) [2–6]. Unfortunately, these studies have been performed with a
small number of genes, and further confirmation is
needed before their clinical application.
The development of DNA microarray technologies is likely to facilitate the generation of novel
diagnostic tools for bladder cancer detection. Thus,
the study of bladder fluids by this technology could
provide a noninvasive diagnostic approach as well
as improve the reliability of current bladder cancer
diagnostic methods. However, application of microarray technology is limited by the requirement of
large amounts of high quality RNA. This requisite is
especially difficult to achieve in some human
samples because of the limited amounts available
and their inherent low RNA quality.
Protocols based on the linear T7dT-based in vitro
transcription method described by Van Gelder and
Eberwine [7,8] can amplify small amounts of mRNA
without significantly distorting the information
content of the sample [9], but they can be sensitive
to RNA degradation. Xiang and colleagues [10]
developed a new procedure that is able to amplify
degraded RNA [10]. This alternative is based on the
use of random nonamer primers modified by the
addition of an upstream T3 promoter sequence
(T3N9) to prime the initial round of reverse transcription.
In this report, we have used the T3N9-based in
vitro transcription method to analyze the suitability
of partially degraded RNA from BW as a sample for
bladder cancer diagnostics with the use of cDNA
microarrays, and we have validated the differential
expression detected by microarrays for four bladder
cancer–related genes on an extended set of BW
samples by quantitative RT-PCR (qRT-PCR).
2.
Methods
2.1.
Samples and RNA preparation
All samples used in this study were collected between June
2003 and January 2005. Individual tumour tissue and bladder
washing samples (T0 and BW0, respectively) were obtained
from a patient with bladder cancer pathologically diagnosed
as pT2 high-grade (HG) transitional cell carcinoma [11,12]
during therapeutical surgery. Four urothelium specimens
from individuals without a history of urothelial cancer were
obtained intrasurgery as control tissue. Tissue samples
(tumour and controls) were immediately frozen after collection in liquid nitrogen and stored at 80 8C until processed.
Fifty BW, 36 from bladder cancer patients (8 pTa low grade [LG],
5 pTa HG, 3 pT1 LG, 5 pT1 HG, 4 pTis, 9 pT2 HG and 2 pT4 HG)
[11,12] and 14 from patients without a history of bladder
cancer were collected. None of the bladder cancer patients
were treated with BCG therapy prior to sample collection.
The hospital ethics committee approved this study and the
patient and controls provided their informed consent before
participating in the study.
For washings, 100 ml of physiologic NaCl solution was
flushed through the bladder about five times via a urethral
catheter with a syringe. Ice-cooled BW samples were mixed
with 1/25 volumes of 0.5 mol/l EDTA, pH 8.0, and were
centrifuged at 1000 g for 10 min. The cell pellets were
resuspended in 1 ml of TRIzol reagent (Invitrogen, Carlsbad,
CA, USA) and frozen at 80 8C until RNA extraction. Tissue and
BW RNAs were extracted with the use of TRIzol reagent
according to the manufacturer’s instructions. Total RNA was
quantified by spectrophotometric analysis at 260 nm.
Equal amounts of intact RNA from T0 and BW0 were
incubated for 15 (T1 and BW1), 30 (T2 and BW2) and 60 (T3 and
BW3) minutes at 80 8C as described [10], with the exception
that RNAse-free water was used instead of base buffer
(Table 1). The four RNAs obtained from the healthy urothelium
specimens were pooled in an equimolar proportion (C0). One
microliter of each intact and partially degraded RNA sample
was analyzed with the use of the Agilent 2100 bioanalyzer
(Agilent Technologies, Waldbronn, Germany) [13] to assess the
degree of degradation (Fig. 1).
2.2.
T3N9-based in vitro amplification and direct
labelling method
Five micrograms of RNA templates were used for RNA
amplification as previously described by Xiang et al. [10,14].
Probes were synthesized by a direct labelling method [15] and
were purified with QIAquick PCR purification columns (Qiagen, Valencia, CA, USA). Labelling efficiency was calculated by
quantification with NanoDrop (NanoDrop Technologies, Wilmington, DE, USA), obtaining between 40–50 pmol/ml of Cy3or Cy5-labelled probes.
2.3.
Array processing and data analysis
Two-colour microarray hybridizations were performed, confronting the four progressively degraded RNAs from each of
the two types of cancer-derived samples against the intact
pool of control bladder RNA, used as common reference in all
hybridizations (Table 1).
Oncochip version 2 glass human cDNA microarrays
produced at the Centro Nacional de Investigaciones Oncolo´gicas (http://grupos.cnio.es/ing/) were used. Fluorescent
images were obtained with the use of the Agilent G2565BA
Microarray Scanner System (Agilent Technologies) and TIFF
images were quantified with the use of the Spot program
(http://experimental.act.cmis.csiro.au/Spot/index.php) under
N/A: not available.
C0 refers to the intact pool of RNA obtained from four healthy bladder tissues (control sample). T0 and BW0 are the intact RNAs isolated from BT and BW samples, respectively, from the same
bladder cancer patient.
b
28S/18S rRNA ratios, % of total 28S and 18S area, and RNA integrity numbers (RINs) are used to quantify the state of RNA degradation.
c
Each specific RNA comparison was performed in duplicate, with dye swapping for a total of 16 hybridization experiments (as a matter of convenience, when we refer to the array name shown in
thetable, we consider the mean values of the array and its dye swap duplicate).
d
Log 2 ratio limits of the 100 most differentially expressed genes of each array.
BW0/C0
BW1/C0
BW2/C0
BW3/C0
36
34
31
23
8.6
7.2
5.8
3.6
24.92
12.17
4.51
0
16.04
12.99
9.13
4.43
1.55
0.94
0.49
0
0
15
30
60
BW0
BW1
BW2
BW3
Cancerous
bladder washing
a
1.68
1.86
1.64
2.27
1.64
1.79
1.64
2.25
0.98
1.06
0.99
1.03
0.97
1.06
1.00
1.00
N/A
N/A
N/A
4
T0
T1
T2
T3
Bladder tumour
0
15
30
60
1.9
1.26
0.75
0
11.47
10.17
7.86
4.43
21.74
12.78
5.9
0
32
31
26
24
T0/C0
T1/C0
T2/C0
T3/C0
–
–
27
8.4
23.44
15.2
1.5
0
C0
Control bladder
28S/18S
ratiob
RNA degradation
time (80 8C) (min)
RNA/array
namea
RNA source
Table 1 – Summary of RNA sample preparation and processing
% of total
18S areab
% of total
28S areab
RINb
RNA yield
(mg)
Samples
cohybridizedc
Log 2 ratio
limitsd
european urology 50 (2006) 1347–1356
1349
the R environment (http://www.r-project.org). The resulting
raw values were filtered and an intensity cutoff was applied,
selecting those points with a foreground median/background
median >3 in at least one channel. The final intensity measure
for each spot was calculated as previously suggested (http://
www.stat.berkeley.edu/users/terry/zarray/html/image.html);
intensity values <150 were considered as background intensities and were converted to 150 for further calculations
(publicly available in Gene Expression Omnibus database;
series GSE3192).
The mean of all considered good spots was obtained as a final
clone intensity measure. Logarithmic ratio (log 2 ratio) was
calculated between both channels (log 2 [R/G]). Of 4269 clones
with at least one valid expression ratio from direct and dye swap
duplicates, only those with an intensity value 400 for at least
one channel were considered (1111 clones). The 100 most
differentially expressed clones of each array were selected to
compare between arrays, and the percentage of overlapping
genes was calculated (Fig. 2A). Common (which appeared
differentially expressed in at least 12 of 16 arrays) and unique
nonoverlapping genes, exclusive of bladder tumour (BT) or BW,
were grouped according to the biologic processes on the basis of
gene ontology (GO) functional annotation (http://david.niaid.
nih.gov/david/version2/index.htm) [16].
Unsupervised hierarchical cluster analysis was performed
for all clones contained in the microarray using unweighted
pair group method with arithmetic averages and Pearson
correlation distance.
2.4.
Quantitative RT-PCR
To validate gene expression data from microarrays (technical
validation of microarray data), 10 of the most differentially
expressed genes (ANXA1, CD74, CDH11, EGR1, HIST1H2AC,
IGF2, ITGA5, KRT20, PPAP2B and VAV3) were tested by qRT-PCR
on intact and partially degraded RNA obtained from BT and
BW. On the other hand, to demonstrate that the differences
between tumoural and control microarrays were mainly due
to the presence of a bladder carcinoma (biologic validation of
the microarray results), we selected four differentially
expressed genes related to bladder cancer according to the
literature (KRT20, IGF2, GSN, and CCL2) [17–20] to be tested in
50 BW (36 cancerous and 14 controls). Two of the selected
genes for the technical validation (KRT20 and IGF2) were also
chosen for the biologic validation since they are involved in
bladder carcinogenesis.
cDNA was synthesized from 1 mg of total RNA with the use
of the High-Capacity cDNA Archive Kit (Applied Biosystems,
Foster City, CA, USA) according to the manufacturer’s
instructions, except that the final volume of the reaction
was 50 ml. The gene GUSB was used as endogenous control.
PCRs were performed with the use of Assays-on-Demand gene
expression products in an ABI Prism 7000 SDS (Applied
Biosystems) according to the manufacturer’s recommendations, except that the final volume of the reaction was 20 ml. All
samples were amplified in duplicate and the CT mean was
obtained for further calculations. Duplicates with SD 0.38
were excluded, and these samples were reamplified. The DDCt
method (ABI Prism 7700 Sequence Detection System User
Bulletin #2: relative quantification of gene expression P/N
1350
european urology 50 (2006) 1347–1356
Fig. 1 – Agilent 2100 bioanalyzer electropherograms from intact and partially degraded RNA samples from bladder tumour (BT),
bladder washing (BW), and control samples. Intact RNAs from tumour and bladder washing samples (T0 and BW0) were heat
shocked at 80 8C for different time periods to obtain three degradation states. The numbers 0, 1, 2, and 3 were assigned to
samples in order of increasing degradation; the same number was chosen for samples with comparable RNA quality.
european urology 50 (2006) 1347–1356
1351
Fig. 2 – Percentage of differentially expressed genes in common between pairs of arrays. (A) Semimatrix comparison
between pairs of arrays of the 100 most differentially expressed genes of each array. The orange-shaded upper and lower
parts of the semimatrix show the percentage of differentially expressed genes identified in common between pairs of BT or
BW arrays, respectively, hybridized with RNA at different degradation states. The white part of the table corresponds to the
percentage of differentially expressed genes identified in common between pairs of tumour and bladder washing arrays. (B)
Unsupervised hierarchical cluster analysis for all clones contained in the microarray, including the respective dye swap
duplicates. DS: dye swapping.
4303859) was used to quantify the relative amount of mRNA in
each tumoural sample in comparison with the C0 sample
(technical validation) or to the mean of the 14 control samples
(biologic validation). Next, 2 DDCt of each gene was transformed into logarithmic scale (log 2 ratio), and comparisons to
the microarray results were performed by means of linear
regression. The analysis of variance test was used to
determine if the expression levels measured by qRT-PCR of
intact and partially degraded RNA were significantly different.
The SPSS software version 12.0 (SPSS Inc, Chicago, IL, USA)
was used for all statistical calculations.
3.
Results
3.1.
Effect of degraded RNA on microarray profiling
Intact RNA from tumour (T0) and bladder washing
(BW0) samples were heat shocked to obtain three
controlled degradation states (Fig. 1 and Table 1).
We cohybridized the four progressively degraded
RNA aliquots from BT and BW with the intact
control bladder RNA. A high percentage of overlapping differentially expressed genes were
detected between BT arrays (85–91%) and between
BW arrays (78–93%) (Fig. 2A). Interestingly, the
percentage of genes in common identified between
the two arrays hybridized with different RNA
degradation states (e.g., BW0 and BW1) was, sometimes, higher than the percentage between dye
swap duplicates of the same array (e.g., BW0 vs
BW0-DS; Fig. 2B).
When we analyzed the suitability of BW for
diagnosing bladder cancer using cDNA microarrays,
we found that the similarity between BW and BT
was relatively high and independent of the RNA
degradation state (52–60%; Fig. 2A). Among the
differentially expressed genes, we detected 48 genes
in common between BW and BT samples (see
Materials and Methods for details). These genes
showed enrichment in ‘‘growth’’ and ‘‘differentiation’’ go categories. On the other hand, we detected
33 and 35 differentially expressed genes exclusive to
BW and BT, respectively. The BW-specific genes
were enriched in ‘‘immune response’’ and ‘‘catabolism,’’ whereas tumour-related genes were, once
again, enriched in ‘‘growth.’’
1352
european urology 50 (2006) 1347–1356
Fig. 3 – Comparison of microarray and quantitative RT-PCR (qRT-PCR) expression data of 10 selected differentially
expressed genes. Positive values indicate overexpression in BT or BW, compared with a pool of healthy bladder tissue
(C0). The down arrows on BW indicate that these samples do not contain enough CDH11 cDNA for an accurate comparison
with the control bladder tissue. Therefore, the stated expression level should be interpreted as the minimum value of
differential expression for BW samples. The numbers 0, 1, 2, and 3 correspond to the RNA degradation states in
dicated in Table 1.
Fig. 4 – Differential expression of four bladder cancer-related genes between tumoral and control BW by qRT-PCR.
Positive values indicate overexpression in tumoral BW compared with the control BW samples. For the control reference
the mean of the 14 BW controls tested was obtained. Samples are arranged by tumoral stage and grade; low grade (LG)
superficial tumours (8 pTa LG and 3 pT1 LG), high grade (HG) superficial tumours (5 pTa HG, 5 pT1 HG, and 4 pTis), and
muscle invasive tumours (9 pT2 and 2 pT4).
european urology 50 (2006) 1347–1356
3.2.
Validation of microarray data using qRT-PCR
We were able to verify the 10 tested genes in BW
samples (100%). In the tumour samples, we could
validate 90% (9 of 10) of tested genes (except
HIST1H2AC) (Fig. 3). Perhaps cross-hybridization
processes that are much more common in microarrays than in qRT-PCR could account for these
differences. The comparison of microarray and qRTPCR results for the 10 selected genes showed a high
correlation between the two methods (r = 0.84,
p < 0.0005), although differences in the magnitude
of change between microarrays and qRT-PCR were
found; 78% of samples had greater gene expression
in qRT-PCR than in microarrays. Otherwise, qRTPCR analysis showed no significant differences in
relative differential expression levels between intact
and partially degraded RNA samples ( p = 0.99).
Gene expression validation of KRT20, GSN, IGF2
and CCL2 by means of qRT-PCR in 36 individual BWs
from bladder cancer patients is shown in Fig. 4. The
percentage of BW samples that confirmed microarray results was 81% for KRT20, 89% for GSN, 64% for
IGF2, and 89% for CCL2.
4.
Discussion
In this study, cDNA microarrays were used to
determine if gene expression profiles derived from
low-quality RNAs obtained from a cancerous BW
sample correlated with those obtained from the
related BT.
Using a controlled RNA degradation procedure,
we obtained different RNA degradation states that
could represent the quality of the vast majority of the
RNA that we generally isolate from BW samples.
Subsequently, we selected the linear T3N9-based in
vitro transcription method to amplify the limiting
RNA quantities usually isolated from bladder fluids.
We found that the quality of the starting RNA was
directly related to the quantity and quality of the RNA
produced by the T3N9-based transcription method
(Table 1). Nevertheless, microarray expression data
did not seem to be practically affected by the initial
RNA quality. Thus, we were able to identify a high
percentage of differentially expressed genes in
common between the different degraded RNA states
of both BT (85–91%) and BW (78–93%) samples
(Fig. 2A). However, even though the RNA degradation
patterns of our RNAs are very similar to those
obtained by other authors who used more physiologic
RNA degradation methods, such as endogenous
tissue ribonucleases [21] or RNAse A [22], it remains
to be shown whether physiologic RNAse degradation
1353
would have an effect on the reproducibility of
measurements on equivalent samples.
Therefore, our data suggest that small amounts of
partially degraded RNA amplified with the T3N9based method may be used in cDNA microarray
studies without a great reduction in the ability to
detect differentially expressed genes. Moreover, it is
also of note that relative gene expression levels
measured by qRT-PCR do not seem to be affected by
RNA degradation either, as previously described [22].
This observation opens up the possibility of performing qRT-PCR studies on inherently degraded
RNA samples such as those extracted from urine,
archival paraffin-embedded specimens, faeces, and
so forth.
In the present study, we also sought to determine
whether gene expression profiles found in BW
reflected those obtained in the related tumour.
The comparison of microarrays hybridized with
both intact RNA samples (T0 vs BW0) yielded an
important percentage of overlapping differentially
expressed genes (59%), indicating the suitability of
the BW sample to reflect tumour gene expression
profiles. It is remarkable that the percentage of
differentially expressed genes in common among all
the microarrays hybridized with different RNA
degradation states is practically constant (52–60%).
On the other hand, although our aim was not to
identify differentially expressed genes in bladder
cancers, we determined that the common differentially expressed genes between BT and BW were
mainly related to cancer (the GO functional annotations were growth and differentiation). It is important to emphasize that some of the genes identified
(e.g., UPK1A, KRT20, IGF2, GSN, ANXA1, CCL2, NRAS,
etc) had been previously described as bladder
cancer-related genes [17–20,23–26] and, interestingly, some of them had already been suggested
as bladder cancer markers in urine or BW [18,27,28].
To confirm some differentially expressed genes
detected in our microarrays in a larger set of BW,
we evaluated KRT20, IGF2, GSN, and CCL2 gene
expressions in 36 individual BW samples by qRTPCR. The concordance of microarray data of a single
patient and qRT-PCR results in an extended series of
BW strongly suggest that our findings were not due
to the particular gene expression profile of the
analyzed patient.
Finally, we focused on gene expression differences between BT and BW. These gene expression
variations may reflect differences in tissue composition, since BW represent primarily urotheliumderived cells, whereas the tumour sample may
contain other cell types such as smooth muscle,
mesenchymal fibroblasts, and endothelial cells [29].
1354
european urology 50 (2006) 1347–1356
We found the growth biologic process category
overrepresented within the list of tumour-specific
genes. Specifically, we identified many genes
involved in nucleic acid metabolism such as EGR1,
ERF, HIST1H1C, or ENTPD5. This observation could be
due to the enrichment of cancer cells in the BT,
compared with the BW sample, which must be
composed by a mix of normal and cancerous cells
producing a dilution effect. When we analyzed
genes expressed exclusively in BW, we found that
functional annotations for immune response and
catabolism were overrepresented. For example, we
have identified genes such as HLA-G, IGLL1, or
IFITM1, which are strongly related with immunity
and defence processes, and genes such as MALT1,
TIMP3, MMP2, or TIMP2, which are involved mainly
in proteolysis and protein metabolism. A possible
explanation for these findings could be the need
for a constant defence of the urothelial mucosa
against external agents, which would be reflected in
the BW. In addition, catabolism processes could be
occurring in the damaged cells present in the
bladder.
5.
Conclusions
In this study, we demonstrated that a small amount
of partially degraded RNA isolated from BW,
processed with the T3N9-based in vitro amplification method, is suitable for inferring gene expression profiles in the related BT by microarray
expression analysis. Our results provide information that could lead to encouraging the development
of a noninvasive method to diagnose and provide a
prognosis in BT, which is based on gene expression
patterns derived from bladder fluid cellular fractions. This alternative diagnostic method could
become available in the future once appropriate
molecular bladder cancer biomarkers are selected.
The next step should be to test if similar results can
be obtained with the use of RNA isolated from urine
samples.
Acknowledgements
This work was supported by Laboratorios Indas S.A.
and by a grant from Fondo de Investigaciones
Sanitarias (FIS04/2630).
The authors would like to thank Manel Ferna´ndez, Lidia Sedano, and David Otero for their
excellent technical assistance and Helena Kruyer
for correction of the manuscript. We are also
grateful to Dr Artur Oliver for his help with the
statistical analysis and to Dr Ferran Algaba (Section
of Pathology) and Dr Humberto Villavicencio
(Department of Urology) for their clinical support.
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Tong D, Schneeberger C, Leodolter S, Zeillinger R. Quantitative determination of gene expression by competitive
Editorial Comment
Morgan Roupreˆt, Urology Department,
Hospital Pitie´-Salpe´trie`re, University Paris VI, France
[email protected]
Bladder cancer is the fourth commonest malignancy amongst men in the western world. The
majority of these tumors are of the superficial
phenotype and most patients (>50%) develop
more than one carcinoma in their lifetime [1].
Approximately 90% of bladder transitional cell
carcinomas are superficial (pTa, pT1, pTis) at
initial diagnosis. The rate of tumor recurrence
ranges 50–80% in the couple of years following
tumor resection and progression to invasive
disease accounts for 15–23% of recurred cases
[1]. Because tumor recurrence, if not detected, may
progress and invade the musculature, early
detection of tumor recurrence is of primary
importance and requires close surveillance for
successful treatment. Follow-up is essentially
based on cystoscopy: the gold-standard method,
despite being invasive and uncomfortable [1].
Non-invasive ancillary methods, less expensive,
with high sensitivity and sensibility would be
of great help in the diagnosis and follow-up
[23]
[24]
[25]
[26]
[27]
[28]
[29]
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reverse transcription-polymerase chain reaction in
degraded RNA samples. Anal Biochem 1997;251:173–7.
Olsburgh J, Harnden P, Weeks R, et al. Uroplakin gene
expression in normal human tissues and locally
advanced bladder cancer. J Pathol 2003;199:41–9.
Sanchez-Carbayo M, Socci ND, Lozano JJ, et al. Gene discovery in bladder cancer progression using cDNA microarrays. Am J Pathol 2003;163:505–16.
Przybojewska B, Jagiello A, Jalmuzna P. H-RAS, K-RAS, and
N-RAS gene activation in human bladder cancers. Cancer
Genet Cytogenet 2000;121:73–7.
Christoph F, Muller M, Schostak M, Soong R, Tabiti K,
Miller K. Quantitative detection of cytokeratin 20 mRNA
expression in bladder carcinoma by real-time reverse
transcriptase-polymerase chain reaction. Urology 2004;
64:157–61.
Eissa S, Kenawy G, Swellam M, El Fadle AA, Abd El-Aal AA,
El Ahmady O. Comparison of cytokeratin 20 RNA
and angiogenin in voided urine samples as diagnostic
tools for bladder carcinoma. Clin Biochem 2004;37:
803–10.
Larsson PC, Beheshti B, Sampson HA, Jewett MA, Shipman
R. Allelic deletion fingerprinting of urine cell sediments in
bladder cancer. Mol Diagn 2001;6:181–8.
Sanchez-Carbayo M, Saint F, Lozano JJ, Viale A, CordonCardo C. Comparison of gene expression profiles in lasermicrodissected, nonembedded, and OCT-embedded
tumor samples by oligonucleotide microarray analysis.
Clin Chem 2003;49:2096–100.
procedures in the daily urological practice. It is
widely accepted that human neoplasms including
bladder cancer arise from the accumulation of
multiple genetic events leading to activation of
proto-oncogenes or inactivation of tumor suppressor genes. New noninvasive methods for the
diagnosis and surveillance of urothelial carcinomas are required to replace cystoscopy. Analysis
of the DNA extracted from exfoliated cells can be
used for noninvasive tumour detection. A noninvasive diagnostic molecular method based on
the detection of loss of heterozygosity (LOH) or
microsatellite instability (MSI) in cells exfoliated
in urine has been reported previously [2]. However, most molecular biomarkers analysed to date
lack sufficient specificity or sensitivity for their
widespread use and require extensive genetic
analysis preventing their routine clinical use
[1,2]. The present article from Mengual et al. is a
study about technical feasibility and ability of
bladder washing to detect or identify early
recurrence in bladder cancer. Although the aim
of the study is not novel, it is quite important to
determine a suitable way to detect urothelial
tumors regarding these new biological tools. The
current results are very interesting because this
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european urology 50 (2006) 1347–1356
team has decided to focus on exclusive RNA
material. The effect of RNA degradation is apparently less important than the type of sample that
is being analyzed. Moreover, gene expression
analysis by microarrays using small amounts of
RNA is becoming more and more popular against
the background of advances and increasing
importance of small-sample acquisition methods
like laser microdissection techniques [3]. The
quality of RNA preparations from such samples
constitutes a frequent issue in this context [3]. If
the results from the next step of the study (i.e.,
RNA isolated from urine samples) are conclusive,
then this should be one of the methods of choice
for these analyses (instead of DNA). One important
issue is to make basic research more understandable for clinicians and urologists and to
involve deeply our community in the ability to use
new, accurate and reliable methods of diagnosis.
These results are a good example and will
probably be of clinical relevance in the daily
urological practice for the nearly future.
References
[1] Chopin DK, Gattegno B. Superficial bladder tumors. Eur
Urol 2002;42:533–41.
[2] Amira N, Mourah S, Rozet F, Teillac P, Fiet J, Aubin P, et al.
Non-invasive molecular detection of bladder cancer
recurrence. Int J Cancer 2002;101:293–7.
[3] Schoor O, Weinschenk T, Hennenlotter J, Corvin S,
Stenzl A, Rammensee HG, et al. Moderate degradation
does not preclude microarray analysis of small amounts
of RNA. Biotechniques 2003;35:1192–201.