Coexistent intraurothelial carcinoma and muscle-invasive urothelial carcinoma of the bladder: clonality and somatic down-regulation of DNA mismatch repair.
ABSTRACT Muscle-invasive urothelial carcinomas are heterogeneous neoplasms for which the clonal relationship with low-grade urothelial dysplasia and carcinomas in situ remains unknown, and both monoclonal and field change models have been proposed. Low-grade dysplasia (18) and carcinoma in situ (12) associated with muscle-invasive urothelial carcinoma were microdissected and topographically analyzed (intraepithelial and invasive superficial and deep to muscularis mucosa) for methylation pattern of androgen receptor alleles, TP53, RB1, WT1, and NF1 microsatellite analysis to assess clonal identity; MLH1 and MSH2 sequencing/immunostaining. Appropriate controls were run. Carcinoma in situ (100%) and invasive urothelial carcinoma (100%) revealed monoclonal patterns, whereas low-grade dysplasia was preferentially polyclonal (80%). Carcinoma in situ showed aneuploid DNA content and more abnormal microsatellites than the corresponding invasive compartments, opposite to low-grade dysplasia. Absent MLH1 protein expression with no gene mutations were identified in carcinoma in situ and nodular-trabecular urothelial carcinoma with high microsatellite abnormalities. Somatic mismatch repair protein down-regulation and the accumulation of tumor suppressor gene microsatellite abnormalities contribute to a molecular evolution for monoclonal carcinoma in situ divergent from coexistent muscle-invasive urothelial carcinoma. Low-grade dysplasia is however unlikely connected with this molecular progression.
- SourceAvailable from: Salvador J Diaz-Cano[Show abstract] [Hide abstract]
ABSTRACT: Tumor heterogeneity is a confusing finding in the assessment of neoplasms, potentially resulting in inaccurate diagnostic, prognostic and predictive tests. This tumor heterogeneity is not always a random and unpredictable phenomenon, whose knowledge helps designing better tests. The biologic reasons for this intratumoral heterogeneity would then be important to understand both the natural history of neoplasms and the selection of test samples for reliable analysis. The main factors contributing to intratumoral heterogeneity inducing gene abnormalities or modifying its expression include: the gradient ischemic level within neoplasms, the action of tumor microenvironment (bidirectional interaction between tumor cells and stroma), mechanisms of intercellular transference of genetic information (exosomes), and differential mechanisms of sequence-independent modifications of genetic material and proteins. The intratumoral heterogeneity is at the origin of tumor progression and it is also the byproduct of the selection process during progression. Any analysis of heterogeneity mechanisms must be integrated within the process of segregation of genetic changes in tumor cells during the clonal expansion and progression of neoplasms. The evaluation of these mechanisms must also consider the redundancy and pleiotropism of molecular pathways, for which appropriate surrogate markers would support the presence or not of heterogeneous genetics and the main mechanisms responsible. This knowledge would constitute a solid scientific background for future therapeutic planning.International Journal of Molecular Sciences 12/2012; 13(2):1951-2011. · 2.34 Impact Factor
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ABSTRACT: PURPOSE: We investigated the relationship between the distribution of the eNOS4a/b polymorphism and the clinical features of superficial bladder cancer. MATERIALS AND METHODS: This study included 201 healthy controls with a mean ± SD age of 62.35 ± 7.96 years and 123 patients with a mean age of 64.03 ± 11.00 years diagnosed with histopathologically confirmed superficial bladder cancer. The eNOS4a/b polymorphism genotype (aa, bb or ab) was identified by polymerase chain reaction. Blood glutathione and plasma malondialdehyde levels were measured by spectrophotometry as an indicator of oxidative stress. We estimated total plasma levels of nitric oxide metabolites using a colorimetric assay kit. RESULTS: There were no significant differences in age or body mass index between patients and controls. Malondialdehyde and nitric oxide metabolite levels were statistically significantly increased (p = 0.000 and 0.024, respectively) and glutathione levels were decreased (p = 0.000) in patients with superficial bladder cancer. The bb genotype of the eNOS4a/b polymorphism is the most frequent one in the Turkish population and the aa genotype was significantly more common in patients with superficial bladder cancer (p = 0.000). Also, the aa plus ab genotype was significantly more common in patients with high grade tumors (p = 0.013) and in those with more progression to muscle invasive disease (p = 0.000). This genotype was also a significant independent risk factor for recurrence after adjusting for smoking status, stage, grade and the presence of carcinoma in situ on logistic regression analyses (OR 3.095, 95% CI 1.21-7.86, p = 0.018). CONCLUSIONS: The current study suggests that a genotype containing the a allele of the eNOS4a/b polymorphism may be a risk factor for bladder cancer. Additionally, patients harboring the aa plus ab genotype are more likely to experience tumor recurrence and progression.The Journal of urology 10/2012; · 3.75 Impact Factor
Coexistent intraurothelial carcinoma and muscle-invasive
urothelial carcinoma of the bladder: clonality and somatic
down-regulation of DNA mismatch repair
Alfredo Blanes MD, PhDa, Javier Rubio MD, PhDa, Juan J. Sanchez-Carrillo MD, PhDa,
Salvador J. Diaz-Cano MD, PhD, FRCPatha,b,⁎
aDepartment of Pathology, University Hospital of Malaga, 29071 Malaga, Spain
bDepartment of Pathology, King's College Hospital and King's College London School of Medicine, SE5 9RS London,
Received 29 July 2008; revised 11 December 2008; accepted 19 December 2008
Carcinoma in situ;
Tumor suppressor genes;
DNA mismatch repair
Summary Muscle-invasive urothelial carcinomas are heterogeneous neoplasms for which the clonal
relationship with low-grade urothelial dysplasia and carcinomas in situ remains unknown, and both
monoclonal and field change models have been proposed. Low-grade dysplasia (18) and carcinoma in
situ (12) associated with muscle-invasive urothelial carcinoma were microdissected and topographically
analyzed (intraepithelial and invasive superficial and deep to muscularis mucosa) for methylation
pattern of androgen receptor alleles, TP53, RB1, WT1, and NF1 microsatellite analysis to assess clonal
identity; MLH1 and MSH2 sequencing/immunostaining. Appropriate controls were run. Carcinoma in
situ (100%) and invasive urothelial carcinoma (100%) revealed monoclonal patterns, whereas low-grade
dysplasia was preferentially polyclonal (80%). Carcinoma in situ showed aneuploid DNA content and
more abnormal microsatellites than the corresponding invasive compartments, opposite to low-grade
dysplasia. Absent MLH1 protein expression with no gene mutations were identified in carcinoma in situ
and nodular-trabecular urothelial carcinoma with high microsatellite abnormalities. Somatic mismatch
repair protein down-regulation and the accumulation of tumor suppressor gene microsatellite
abnormalities contribute to a molecular evolution for monoclonal carcinoma in situ divergent from
coexistent muscle-invasive urothelial carcinoma. Low-grade dysplasia is however unlikely connected
with this molecular progression.
© 2009 Elsevier Inc. All rights reserved.
Urothelial dysplasia is assumed to be the putative precursor
of urothelial carcinoma (UCC) and confers a significant risk for
the development of carcinoma in situ (CIS) and invasive UCC,
as reported for intraepithelial breast or melanocytic lesions .
Although the accumulated molecular data indicate that most
recurrent and multiple tumors are monoclonal, the controversial
Presented in part as abstracts in the meetings of the United States and
Canadian Academy of Pathology, Atlanta, GA, 2001; San Antonio, TX,
2005; and Denver, CO, 2008; and Pathological Society of Great Britain and
Ireland, Maastricht, Netherland, 2001.
⁎Corresponding author. Department of Histopathology, King's College
Hospital, SE5 9RS London, United Kingdom.
E-mail address: email@example.com (S. J. Diaz-Cano).
0046-8177/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Human Pathology (2009) 40, 988–997
definitions of flat lesions with atypia, that is, reactive atypia,
atypia of unknown significance, low-grade urothelial dysplasia
(LGUD), and high-grade urothelial dysplasia-CIS , have
contributed to create confusion for coexistent lesions . In
addition, the concept of tumor progression is not consistently
used in bladder pathologic examination , being a cytologic
progression documented in urothelial dysplasias but with a
different topography for LGUD and CIS. This supports the
multifocal distribution of low-grade UCC without proving a
clonal identity of LGUD and CIS to sustain the sequence
LGUD —N CIS.
The clonality status of multifocal bladder tumors is still
controversially discussed with experimental evidence for both
monoclonality and field cancerization. Early stage urothelial
neoplasms have shown chromosome 9 deletions and FGFR3
mutations [5,6], the same genetic alteration being observed in
coexistent low-grade papillary superficial UCCs and histologi-
cally normal urothelium, whereas 17p13 hemizygosity was
observed in a minority of urothelial hyperplasias and papillary
tumors but not in normal urothelium. This genetic profile
suggests that the earliest molecular alterations in the pathogen-
esis of low-grade UCC involve p16/CDKI2 but not TP53 even
in histologically normal areas, but this study does not analyze
Clusters of discontinuous deletedsegmentsoftumorsuppressor
gene loci on chromosomes 13q14 and 17p13 have been
associated with clonal expansion of in situ bladder preneoplasia
using single nucleotide polymorphisms . The clonal relation-
ships between LGUD and CIS associated with muscle-invasive
UCC have not been addressed. Although bladder UCC
expression of mismatch repair (MMR) proteins contributes to
the development of a subset of UCC [12,13].
The relationship (linear versus divergent) between intraur-
othelial and invasive compartments (superficial and deep) has
not been topographically analyzed in muscle-invasive UCC
showing these 3 compartments. This study evaluates in each
topographic compartment as follows: clonality, MMR protein
expression/sequencing, and loss of heterozygosity (LOH)-
microsatellite profile of tumor suppressor genes (TSG)
controlling G1-S transition (TP53, RB1), RAS pathway (NF1),
and development (WT1). All these analyses have not been
performed simultaneously in a series of muscle-invasive and
intraurothelial lesions and will inform on both monoclonal/field
change models in muscle-invasive UCC and tumor progression
from the perspective of the accumulation of genetic alterations
in tumor suppressor genes.
2. Materials and methods
2.1. Case selection and sampling
We reviewed the initial transurethral resection biopsy of
pT2a/b UCC with no special differentiation of the bladder
diagnosed in women (44 cases), treated with cystectomy and
lymphadenectomy from 3 reference hospitals (1990-1992,
median follow-up 60 months). Transurethral resection
biopsies were selected because they provided better cellular
preservation, and the results for molecular tests are more
reliable as they show lower frequency of artifacts [14,15].
Intraurothelial neoplastic lesions were classified according to
the World Health Organization system in low-grade
dysplasia (LGUD, 11 patients) and carcinoma in situ (CIS,
7 patients) , both coexisting in 3 patients. To add power to
the clonality analysis, a combined approach of X chromo-
some inactivation and tumor suppressor gene microsatellite
profile was selected. The X chromosome inactivation
requires samples from female that represent the initial
patient selection; to extend the analysis, cases from male
were selected (28, which revealed LGUD in 7 patients and
CIS in 5 patients, coexistent lesions in 2), the selection
criteria then included matching cases by age and conven-
tional histologic features of the muscle-invasive component
to avoid any biases from those aspects. The cases were
classified independently by 2 pathologists (AB, SDC). In
case of grading disagreement, the lesions were discussed
during simultaneous inspection before final categorization.
Reproducibility data were not recorded.
All surgical specimens were completely embedded for
histopathologic diagnosis. The topographic compartment
limit was the muscularis mucosa (superficial and deep to
the muscularis mucosa), being the same areas analyzed in
each study [16-18]. The muscularis mucosae was selected as
limit because tumors invasive to this level had shown a
much better 5-year survival than tumors invasive through the
level of the muscularis mucosae, which showed survival
comparable with patients with tumors invasive of the
muscularis propria . This protocol was approved by
the Hospital Research Board and Ethical Committee and
complied with their requirements.
2.2. Clonality assay and TSG microsatellite analysis
DNA was extracted from the most cellular areas of
intraurothelial, superficial, and deep compartments, after
microdissecting at least 100 cells (approximately 0.4 mm2,
laser capture; Arturus, Sunnyvale, CA) from two 20-μm
unstained paraffin sections/compartment. Appropriate tissue
controls (histologically normal urothelium, stroma from the
lamina propria, and smooth muscle) and quality-assurance
controls (sensitivity, specificity, positive, and negative) were
run for each test [14,15,20].
DNA was extracted using a modified phenol-chloroform
protocol, precipitated with ice-cold absolute ethanol, and
resuspended in 10 μL of Tris-HCl buffer pH 8.4 . DNA
was then used for polymerase chain reaction (PCR) amplifica-
tion of TSG intron microsatellites and the hypervariable CAG
repeat in the first exon of the human androgen receptor (see
Table 1 for primer sequences and cycling conditions), using
989 MMR and Microsatellites in VCC
HhaI-undigested and digested samples for the X chromosome
inactivation assay that contained XhoI-linearized φX174-RII
phage (Gibco-BRL, Gaithersburg, MD) as mimicker of
digestion completion checked by gel electrophoresis (Table 1)
[16,17,20-22]. The tests were run in a Perkin-Elmer thermal
cycler model 480 (Perkin-Elmer, Norwalk, CT). The whole 10-
μL PCR volume was electrophoresed into 8% denaturing
gradient polyacrylamide gels; dried gels were put inside
developing cassettes containing one intensifying screen and
preflashed films (Kodak XAR) [17,23]. The radiographs were
developed using an automated processor Kodak-Omat 100
(Kodak Co, Rochester, NY).
Interpretation and inclusion criteria included [14,17,20-22]
the following: (a) allelic imbalance was densitometrically
evaluated (EC model 910 optical densitometer, EC Appara-
tus Corporation, St Petersburg, FL), considering evidence of
LOH only allele ratios 4:1 or greater in any TSG; otherwise,
retention of heterozygosity was assigned [17,22]. This ratio
would represent 80% of clonal cells in the sample and was
used to increase the detection specificity [20,21,24]. (b)
Additional allele bands present in tumor samples but not in
the corresponding controls were considered evidence of
somatic microsatellite abnormality by PCR/denaturing
gradient gel electrophoresis .
2.3. DNA sequencing
All microsatellite extrabands were cut from gels, and
DNA was purified using a QIA quick gel extraction kit
(Qiagen, West Sussex, UK). The amplified product was
diluted 20-fold in TE buffer, and 1-μL of the diluted reaction
product was subjected to a second round of PCR amplifica-
tion using the appropriate primers for 30 cycles under the
above conditions. Normal and extrabands from tumor-
derived samples were PCR amplified along with the
corresponding controls using a high-fidelity polymerase,
Platinum PFX (Life Technologies). PCR products were
directly sequenced after purification (QIAquick PCR
purification kit, Qiagen). All sequencing was performed on
an ABI Prism 3700 automated DNA analyzer, and the
sequence data were analyzed using the program Sequencher
Primer sequences and PCR cycling conditions
Primer sequencesRepeats/PCR product PCR cycling conditions
⁎AR alleles were amplified using
“hot start” protocol, 0.3 μmol/L of
each primer, and 200 μmol/L of
each dNTP (including 7-deaza-dGTP
instead of dGTP) (Boehringer-Mannheim,
Indianapolis, IN), completing 28 cycles
with an annealing temperature of 55°C.
The amplicon was internally labeled
with 0.3 μCi α[32P]-dTTP (800 Ci/mmol,
10 mCi/mL; New England Nucleotide,
‡TSG polymorphic regions were amplified
using 0.25 μmol/L of each primer,
50 μmol/L of each dNTP
(Boehringer-Mannheim, Indianapolis, IN),
and internally labeled with 0.3 μCi
α[32P]-dCTP (3000 Ci/mmol, 10 mCi/mL;
New England Nucleotide, Boston, MA).
The annealing temperature was 55°C
(except for NF1, 52°C), and the number
of cycles was experimentally optimized
5′-CCG AGG AGC TTT CCA GAATC-3′
5′-TAC GAT GGG CTT GGG GAG AA-3′
TP53(1)-F‡5′-AGG GAT ACT ATT CAG CCC-3′
TP53(1)-R‡5′-ACT GCC ACT CCT TGC CCC ATT C-3′
TP53(2)-F‡5′-GAATCC GGG AGG AGG TTG-3′
TP53(2)-R‡5′-AAC AGC TCC TTT AAT GGC AG-3′
5′-CTC CTC CCC TAC TTA CTT GT-3′
5′-AAT TAA CAA GGT GTG GTG GTA CAC G-3′
5′-AAT GAG ACT TAC TGG GTG AGG-3′
5′-TTA CAC AGT AAT TTC AAG CAA CGG-3′
5′-CAG AGC AAG ACC CTG TCT-3′
5′-CTC CTA ACATTT ATT AAC CTT A-3′
CA/approximately 144 bp
Abbreviations: F, forward; R, reverse.
All reactions were run in duplicate using 1.5 mmol/L of MgCl2and 1 μL of template, as well as long denaturation (4 minutes) and expansion (90 seconds) in
the first 3 cycles. The appropriate PCR primers were designed using Genrunner software (version 3.02; Hastings Software Inc., Hudson, NY).
990A. Blanes et al.
(Gene Codes Corporation, Ann Arbor, MI), which reverses
and complements the antisense strand. All mutations were
confirmed by sequencing in both directions and indicated by
an “N” in the sequencing chromatogram.
MLH1/MSH2 exons were completely sequenced in cases
with microsatellite abnormalities in at least 40% loci and/or
completeloss ofmlh1/msh2 immunoreactivity,aswellasina
sample of mlh1/msh2 immunoreactive UCC  as controls.
2.4. Immunohistochemical detection of TP53,
MLH1, and MSH2
The sections were mounted on positively charged slides
(Superfrost Plus, Fisher Scientific, Fair Lawn, NJ), baked at
60°C for 2 hours, and processed as described [16,17,25]. After
routine dewaxing and rehydration, endogenous peroxidase
quenching, and antigen heat retrieval (pressure cooker, citrate
buffer [10 mmol/L], for all antibodies), the slides were
transferred to a moist chamber. Nonspecific binding was
blocked with polyclonal horse serum, and sections were
incubated with monoclonal primary antibodies (overnight,
4°C) as follows: 2 μg/mL of p53 DO-7, Calbiochem,
Cambridge, MA; hMLH1 clones G168 728 and G168-15,
BD Pharmingen Biosciences, Oxford, UK; hMSH2 clone
FE11, Oncogene Research (Merck Chemicals Ltd., Notting-
ham, UK). Then sections were serially incubated with
biotinylated antimouse antibody and peroxidase-labeled avi-
din-biotin complex. The reaction was developed under
microscopic control, using 3,3′-diaminobenzidine tetrahy-
drochloride with 0.3% H2O2as chromogen (Sigma Co, St
Louis, MO), and the sections were counterstained with
(omitting the primary antibody) controls were simultaneously
run. Basal cells of the unaffected urothelial mucosa were used
as internal positive controls for mlh1 and msh2.
2.5. Nuclear DNA quantification by slide cytometry
Feulgen-stained sections were used for DNA quantifica-
tion . The densitometric evaluation was performed with
the cell analysis system model 200 and the quantitative DNA
analysis software package (Becton Dickinson, Oxford, UK).
At least 300 complete, nonoverlapping, and focused nuclei
(or the whole lesion if smaller) were measured in every case,
beginning in the most cellular area until completion in
consecutive high-power fields (HPFs).
replacing the muscularis propria) but not with infiltrative UCC (small tumor nests/thin cords embedded in a prominent desmoplastic reaction,
dissecting the smooth muscle fibers) (hematoxylin-eosin, original magnification ×400).
Coexistent urothelial CIS was associated with nodular-trabecular UCC (sheets of neoplastic cells with minimal stromal reaction,
991 MMR and Microsatellites in VCC
External staining calibration was carried out with
complete rat hepatocytes (Becton-Dickinson; one slide per
staining holder) to normalize the internal controls (lympho-
cytes and histologically normal urothelial cells present in the
same tissue section), used for setting the G0/G1cell limits
and calculating the DNA index of each G0/G1peak (N10% of
measured cells with evidence of G2 + M cells) .
Proliferation rate (PR = S + G2+ M-phases fraction) was
calculated from the DNA histogram by subtracting the
number of cells within G0/G1limits from the total number of
measured cells and expressed as percentage [26,27].
External diploid controls were used to determine DNA
indices (lymphocytes from reactive lymph nodes) and to
standardize the nuclear area/DNA content analysis (normal
transitional cells) .
2.6. Tumor infiltration pattern, grading, and
mitotic figure counting
The infiltration pattern was evaluated in deep compart-
ments, classifying the tumor by the predominant pattern
(N50%) in nodular-trabecular and infiltrative . The
histologic grading evaluated architectural features, nuclear
grade, and mitotic figure (MF) counting . MFs were
screened in 50 HPF/compartment (7.140 mm2) or the whole
tumor if smaller (3 superficial and 6 deep compartments),
beginning in the most cellular area . Both the number of
positive nuclei/HPF and the number of neoplastic cells
intercepted by the microscope field diameter (n) were
registered, the latter to estimate the number of neoplastic
cells/HPF (N = [nπ/4]2) ; results were expressed per
1000 cells, calculating average and SD per compartment and
patient. Tumors were graded by 3 independent observers (JR,
AB, and SJD-C), being the tumor discussed during
simultaneous inspection before final categorization in case
of disagreement. Reproducibility data were not recorded.
2.7. Quantification of positive nuclei and
group, beginning in the most cellular area. The number of
positive nuclei was expressed per HPF and per 1000 tumor
cells, calculating average and SD for each pathologic condition
suppressor gene microsatellite (TSG MS) abnormalities in LGUD and CIS. Abbreviations: ROH, retention of heterozygosity (green cell);
LOH/SNP, loss of heterozygosity/single nucleotide polymorphism (red cell); NI, noninformative (gray cell); TP53, tumor protein p53; RB1,
retinoblastoma; WT1, Wilms tumor 1; NF1, neurofibromatosis 1.
Clonality and microsatellite profile in bladder intraurothelial lesions associated with muscle-invasive UCC. Frequency of tumor
992A. Blanes et al.
established at the positive control in each staining batch. Only
nuclei with staining features similar to those of their
corresponding positive control were considered positive for
Categorized variables were tested using Fisher exact tests
and quantitative variables using Student t tests (if normally
distributed) and nonparametric tests (Mann-Whitney for 2-
group comparisons and Kruskal-Wallis for N2-group com-
parisons). Differences were considered significant if P b.05
in 2-tailed distributions.
CIS was identified in patients with nodular-trabecular UCC
(Fig. 1) and revealed more abnormal TSG loci than the
corresponding invasive compartment (10/12 patients), TP53
loci being involved in all patients (Fig. 2) with expression of
abnormal p53 protein. CIS showed either an additional TSG
locus involved (8 patients; TP53 in 5, RB1 in 2, and WT1 in 1)
or a combined pattern of superficial and deep compartments (2
patients). The other 2 cases showed TP53 abnormality only or
different microsatellite abnormalities at the same loci in
intraurothelial and invasive compartments. In contrast, LGUD
revealed LOH in 2 patients, one at RB1 (monoclonal
methylation of androgen receptor alleles) and the second at
WT1-NF1 loci (polyclonal pattern), respectively (Fig. 2). CIS
(6; 100%), invasive UCC (13; 100%), and LGUD (2; 20%)
from informative females revealed unbalanced methylation
pattern of androgen receptor alleles, whereas polyclonal
patterns were observed in LGUD only (8; 80%; Figs. 2
and 3). Discordant pattern of AR allele was observed in one
case, the larger allele being methylated in LGUD and the
smaller allele in CIS-invasive UCC. The UCC microsatellite
profile of superficial and deep compartments was proven
statistically different from CIS profile at TP53 locus only (P =
0.042; Fig. 4), showing similar topographic heterogeneity in
UCC invasive compartments, regardless of the presence or
absence of CIS.
superficial [sup] and deep compartments) (panel A). Representative gels of the methylation allele pattern of androgen receptor from CIS
(monoclonal, TCC1-CIS) and LGUD (monoclonal, TCC7-LGUD, and polyclonal, TCC2-LGUD) (panel B). Mismatch protein expression.
Nuclear mlh1 and msh2 expression is demonstrated in UCC with no microsatellite abnormalities and at least one of these proteins was absent
(in particular mlh1) in UCC with microsatellite abnormalities. MLH1 and MSH2 exon sequencing. Normal sequence is demonstrated for these
genes, regardless of the microsatellite pattern.
TSG microsatellite pattern in carcinoma in situ (CIS, all monoclonal), LGUD (mainly polyclonal), and muscle-invasive UCC (from
993 MMR and Microsatellites in VCC
Nodular-trabecular UCCs were more frequently aneuploid
(27/28; 96%) and high grade (26/28; 93%) than infiltrative
UCCs (9/16, 56%, and 12/16, 75%, respectively). Nodular-
trabecular UCCs and superficial compartments showed sig-
nificantly higher values for both mitotic figure counting and
proliferation rate (Table 2).The number of diploid (6 cases) and
low-grade (5 cases) UCCs precluded any statistical compar-
abnormal loci than infiltrative UCCs (Fig. 2; P = .0001).
Discordant genetic patterns by tumor compartments were
observed in only 2 infiltrative UCCs precluding any statistical
assessment, but all showed nuclear TP53 expression and more
LOH/single nucleotide polymorphisms (SNPs) in the deep
compartment (WT1 LOH/SNP in 1 and NF1 LOH/SNP in 1).
Immunostaining for mlh1/msh2 revealed statistically sig-
nificant reduction of at least one of the proteins (especially
mlh1) in CIS and the deep compartment of trabecular UCC
with 2 or more TSG microsatellite abnormalities (Fig. 3). No
significant difference was observed in the mlh1/msh2 immu-
noexpression in UCC with less than 2 TSG genetic
abnormalities (MS stable or MS instable-low) but revealed
deficient MMR system at the deep compartment. Normal
MLH1/MSH2 exons sequences were observed in all UCC
analyzed, regardless of immunoexpression and microsatellite
status (Fig. 3).
Microsatellite analysis of TSG supports that coexistent
CIS and muscle-invasive UCC evolves independently,
contributing to intratumoral heterogeneity, despite having
a common progenitor (monoclonal proliferation). The
somatic MMR protein down-regulation contributes to the
different in UCC with dysplasia compared with UCC without dysplasia. Microsatellite abnormalities were more frequent in superficial
compartments (RB1, P b .001; NF1, P = .004) and deep compartments (RB1, P = .003) of UCC without dysplasia. Different patterns were
noted in the superficial and deep compartments from a given patient in 5 patients with urothelial dysplasia (20%). This topographic genetic
heterogeneity was due to the presence of additional abnormalities in superficial compartments (NF1, 1 patient) or deep compartments (TP53, 2
patients; RB1, 1 patient) or discordant pattern (1 patient). Frequency of TSG microsatellite abnormalities in superficial and deep compartments
of muscle-invasive UCC. Abbreviations: ROH, retention of heterozygosity (green cell); LOH/SNP, loss of heterozygosity/single nucleotide
polymorphism (red cell); NI, noninformative (gray cell); TP53, tumor protein p53; RB1, retinoblastoma; WT1, Wilms tumor 1; NF1,
neurofibromatosis 1; S, superficial; D, deep.
Clonality and microsatellite profile of muscle-invasive UCC by topographic compartments. The genetic pattern was statistically
994 A. Blanes et al.
accumulation of genetic alterations, which suggests an
independent evolution rather than a precancerous lesion/
early neoplasm. LGUD is mainly a polyclonal lesion
revealing low incidence of TSG microsatellite abnormalities,
suggestive of a nonneoplastic condition.
A linear progression model for UCC would expect to
find progressive accumulation of genetic alterations in the
transition intraurothelial-superficial invasive-deep invasive
components of UCC. However, this gradient is not found.
CIS displays microsatellite alterations similar to UCC deep
compartments [1,16,17] but with more genetic abnormal-
ities in CIS (Figs. 2,4,5). This and the differential TSG
microsatellite pattern of UCC superficial compartments
(NF1-defective) [16,17] do not support the sequence
coexistent CIS—Nsuperficial UCC—Ndeep UCC .
Several intraepithelial foci show more alterations than
matched invasive foci, suggesting a more extensive genetic
evolution for the former and supporting multifocality and
independent clonal evolution of these coexistent carcino-
mas [30,31]. The accumulation of genetic abnormalities in
coexistent CIS is consistent with an independent progres-
sion of bladder carcinoma (Fig. 2) [32-37]. Genetic
alterations centered around RB1 may represent an incipient
event in bladder neoplasia. However, the inactivation of
RB1 occurred later and was associated with the onset of
severe dysplasia/carcinoma in situ . There is also
evidence for the presence of critical alternative candidate
genes mapping to the 13q14 region that are involved in
(superficial versus deep)
Kinetic features of muscle-invasive UCC by infiltration patterns (nodular-trabecular versus infiltrative) and tumor compartments
Nodular-trabecular pT2a/b UCCInfiltrative pT2a/b UCC Significance
9.0 ± 5.1
33.74 ± 7.60
4.1 ± 3.1
19.69 ± 6.15
5.9 ± 3.5
26.46 ± 8.36
2.0 ± 1.8
14.57 ± 4.58
P = .012
P = .009
growth pattern (trabecular-nodular with coexistent CIS versus infiltrative) and TSG (NF1/RB1/TP53) regulation of the G1-S transition
(interstitial DNA loss versus no DNA loss).
Muscle-invasive UCC can evolve through pathways with and without microsatellite abnormalities that correlate with the invasive
995MMR and Microsatellites in VCC
clonal expansion of neoplasia within the bladder ante-
cedent to the inactivation of the RB1 gene . Finally, we
performed high-resolution mapping using single nucleotide
polymorphism markers within one region on chromosome
13q14, containing the model tumor suppressor gene RB1,
and defined a minimal deleted region associated with
clonal expansion of in situ neoplasia. These analyses
provided new insights on the involvement of several
noncoding sequences mapping to the region and identified
novel target genes, termed forerunner (FR) genes, involved
in early phases of cancer development . In addition,
the invasive compartment microsatellite pattern of UCC
with intraurothelial lesions revealed a significant decrease
of RB1 and NF1 abnormalities (Fig. 4), which correlated
with a nodular-trabecular pattern and high cellular turnover
, proving the topographic genetic heterogeneity of
muscle-invasive UCC [16,17].
LGUD shows much lower incidence of genetic abnorm-
alities, making the direct connection with the linear
progression unlikely. Although a clonal relationship has
been suggested by LOH and/or comparative genomic
hybridization analyses, supporting the hypothesis that flat
urothelial hyperplasias can display many genetic alterations
commonly found in bladder cancer , the kinetic features
of LGUD makes unlikely this progression. The combined
LGUD showed low incidence of TSG microsatellite
abnormalities, no TP53 alterations, and polyclonal patterns.
These results support the existence of 2 transformation
pathways for bladder UCC, TP53 alterations in high-grade
UCC (frequently muscle-invasive) and p16 in low-grade
UCC (often pT1) . Our results also disprove the clonal
identity of coexistent LGUD and CIS, and their dissimilar
—Nco-existent CIS. The hypotheses of tumor evolution and
by deletion-independent clonality studies as X chromosomal
are clinically important features of urothelial carcinomas of
the urinary bladder. Combination of molecular data with
histopathologic bladder mapping suggested a monoclonal
development of the multifocal lesions mostly via intraur-
othelial migration. Recent molecular genetic studies have
suggested that multifocal urothelial carcinomas are mono-
MMR protein down-regulation, normal MLH1/MSH2
sequences, and microsatellite abnormalities characterized CIS
(12 cases, P = .0053) and nodular-trabecular UCC.
Microsatellite profiles of bladder UCC have shown
infrequent instability [9-11], which can be an independent
prognostic marker for assessing risk of recurrence in
superficial tumors irrespective of the grade . Likewise,
reduced expression of the MMR proteins may have an
important contribution in the development of a subset of
UCC and is a potentially useful prognostic marker [12,13],
in particular for upper urinary tract tumors . However,
the pattern of microsatellite instability depends on the
location and elevated microsatellite alterations at select
tetranucleotides being reported more frequently in bladder
. Our results add other type of microsatellite abnorm-
alities (extrabands due to single nucleotide substitutions) in
muscle-invasive UCC revealing solid-trabecular growth
pattern and coexistent CIS. MMR gene inactivation (by
either mutation or protein down-regulation) leads to mutation
accumulation and molecular progression not necessarily
independent from chromosomal instability .
In conclusion, TSG microsatellite patterns support a
nonlinear and independent genetic evolution of coexistent
CIS and invasive UCC (Fig. 5). The combined assessment of
clonal identity (LOH of tumor suppressor genes and X-
chromosome inactivation) and molecular progression (defined
transmission of genetic alterations to descendant cells. Somatic
MMR protein down-regulation, the accumulation of TSG
microsatellite abnormalities, and monoclonal pattern suggest a
divergent molecular evolution for CIS and coexistent muscle-
invasive UCC (in particular those with trabecular growth
pattern). UCC with infiltrative pattern should follow alternative
pathways and LGUD are most likely unrelated with the
mechanisms of somatic down-regulation of mismatch repair
system. In contrast, LGUD shows polyclonal pattern and low
incidence of TSG microsatellite abnormalities, suggestive of a
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