Download Bladder Cancer: Allelic Deletions at and around the Retinoblastoma

610 Vol. 6, 610 – 615, February 2000
Clinical Cancer Research
Bladder Cancer: Allelic Deletions at and around the Retinoblastoma
Tumor Suppressor Gene in Relation to Stage and Grade1
Takashi Wada, Jari Louhelainen,
Kari Hemminki, Jan Adolfsson, Hans Wijkström,
Ulf Norming, Erik Borgström, Johan Hansson,
Bengt Sandstedt, and Gunnar Steineck2
Department of Biosciences at Novum, Karolinska Institute, 141 57
Huddinge [T. W., J. L., K. H.]; Department of Urology, Huddinge
University Hospital, 141 86 Huddinge [J. A., U. N., H. W.];
Department of Urology, Karolinska Hospital, 104 01 Stockholm
[E. B.]; and Clinical Epidemiology [T. W., G. S.] and Clinical
Oncology [J. H.], Department of Oncology-Pathology, Karolinska
Hospital, 171 76 Stockholm, Sweden
ABSTRACT
Inhibition of the retinoblastoma tumor suppressor gene
(RB) is probably important in the pathogenesis of urinary
bladder cancer. Little information is available concerning
allelic loss on 13q11 to 13q32 and its relation to grade and
stage. In a population-based study, freshly frozen tissue was
collected from all new cases of urinary bladder cancer in the
Stockholm region during 1995–1996. Here we report the
occurrence of loss of heterozygosity (LOH) at seven sites in
13q11 to 13q32 as analyzed in 236 cases by a fluorescent
multiplex PCR-based on tumor DNA and peripheral blood.
For each site, about 30% of the cases were not informative
because of homozygosity. Replication errors were detected
in 4% (17 cases). LOH was found in 21 (at 13q11–12.1) to
32% (at 13q14.3 in RB) of the informative cases. A correlation was found between the prevalence of LOH at all observed loci and stage and grade, respectively, and it was
statistically significant for 13q14.3. LOH at RB was found in
Ta as well as grade 1 tumors. Also, a statistically significant
correlation was found between the number of loci with LOH
at 13q and tumor stage and grade, respectively. Typically an
altered RB function is related to the expected clinical course
of urinary bladder cancer, but allelic loss including the gene
also occurs in low grade and low stage tumors. An altered
RB function probably is not necessary for a malignant transformation of urothelial cells. The causal direction of the
relation between the quantity of the deleted DNA and tumor
aggressiveness is not clear.
Received 12/7/98; revised 11/1/99; accepted 11/15/99.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
Supported by the Swedish Cancer Society and the Stockholm Cancer
Foundation (Cancerföreningen i Stockholm).
2
To whom requests for reprints should be addressed, at Clinical Epidemiology, Department of Oncology-Pathology, Karolinska Hospital,
171 76 Stockholm Sweden. Phone: ⫹46-8-517-75080; Fax: ⫹46-8347859; E-mail: [email protected].
INTRODUCTION
An altered function of one or several of the tumor suppressor genes p16, p53, and RB3 is part of the pathogenesis in many
cases of urinary bladder cancer; allelic loss, mutation, or methylation of at least one of these genes have been found in all
series investigated (1–5). The micropathology of the disease is
further outlined by reports of allelic loss at a number of sites
other than 9p (p16), 13q (RB), and 17p (p53), including chromosomes 3p, 4p, 8p, 9q, and 11p (6 –10). It has been argued that
the observation of all these changes indicates that p16, p53, and
RB constitute just a few of a plenitude genes that are important,
but also— on the contrary—that many changes purely reflect a
genetic instability of malignant cells. Any suggested pathogenetic pathway rests on sparse data. Although the simplicity of
the “two-hit” model for tumor development built on inherited
RB mutations is attractive, more events than two typically are
suggested as important when tumor formation is modeled (11).
The increased risk of the disease in families with a germline RB mutation and the occurrence of genetic changes corresponding to RB (detected by varying techniques) display the
relevance of RB inhibition to urinary bladder cancer pathogenesis (4, 5, 12–15). Advanced stage has been related to genetic
changes indicating RB inhibition, but it is unclear if the tumor
aggressiveness depends on the biology of the gene by itself or,
for example, concurrent genetic changes. RB codes for a Mr
110,000 nuclear phosphoprotein that probably regulates the cell
cycle at the G1-S transition (16 –19). Apart from retinoblastoma
and urinary bladder cancer, RB seems to be important in breast,
colorectal, and prostate cancer (20 –22).
Possibly a large unselected population-based cohort can
add validity when one elucidates pathogenesis. In Stockholm,
we have collected a consecutive population-based series of more
than 600 newly diagnosed cases of bladder cancer during 1995–
1996. In the present analysis of 236 patients, we describe allelic
loss at different loci between 13q11 and 13q32, that is, from a
broad region adjacent to, and including, RB.
MATERIALS AND METHODS
Patients and Tissue. Through a collaboration between
urologists in Stockholm, all newly diagnosed cases of urinary
bladder cancer were identified during 1995–1996. Before transurethral resection, four tissue samples were taken by cold-cup
biopsy and freshly frozen at ⫺80°C. The frozen tissue was cut
in to about 5-␮m-thick sections, and the first and last sections
were stained and examined for tumor content. A sample with at
least 70% tumor content was required for inclusion in the
present analysis. Tumor DNA was extracted by a previously
described method (23).
3
The abbreviations used are: RB, retinoblastoma tumor suppressor
gene; LOH, loss of heterozygosity.
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Clinical Cancer Research 611
Blood. Venous blood from each patient was collected in
EDTA tubes and frozen. Leukocyte DNA was prepared from
200 ␮l of whole blood, adding 800 ␮l freshly 170 mM ammonium chloride for 20 min of vortex mixing. White cells were
collected by centrifugation at 3000 rpm for 2 min. The pellet
was washed with 300 ␮l of 10 mM NaC1/10 mM EDTA three
times to remove hemoglobin. The pellet was resuspended in 500
␮l of 50 mM NaOH and incubated for 20 min at 100°C. Afterward, 100 ␮l of 1 M Tris-HCl (pH 7.5) was added to adjust the
pH to be neutral. Cell debris was removed from the solution by
centrifugation, and leukocyte DNA was stored at ⫺20 or
⫺80°C.
PCR Amplification. We used seven microsatellites with
3- or 4-bp repeat motifs. Primer sequences for these markers
were obtained from the Genome Database.4 RB1.20, located 54
bp from the 3⬘ end of exon 20 at 13q14.3, was used as an
internal RB gene marker (24). PCRs were carried out using
Perkin-Elmer DNA cycler (Norwalk, CT) in a 10-␮l reaction
volume containing 0.2 mM of each primer (one of the primers
labeled with colors FAM, HEX, or TET), 2.00 mM MgC12, 0.20
mM dNTP, 1 unit of Taq polymerase, and about 10 ng of
genomic DNA. The DNA was amplified for 32–36 cycles at
94°C during 45 s, at 55°C during 60 s, and at 72°C during 45 s.
Multiplex PCR. The Multiplex PCR method was the
same as described above with the exception that 2–7 primer
pairs were added in the same PCR tube. Primers were selected
on the basis that the resultant PCR products could be distinguished by color and by size range without overlapping fragments.
PAGE. PCR products were analyzed on 4% polyacrylamide denaturing gels in 1⫻ TBE buffer in a model 377 automated fluorescent DNA sequencer (Applied Biosystems, Foster
City, CA), which has four-color detection systems. One microliter of each PCR product was resuspended using 3.5 ␮l of
loading solution (2.5 ␮l formamide, 0.5 ␮l Blue Dextran (50 mM
EDTA with 50 mg/ml Blue Dextran), and 0.5 ␮l GeneScan Size
Standard: GS500). This mix was denatured at 95°C during 5
min and 1.5 ␮l was loaded into each well. The gel was run for
2 h at 40 W and 52°C.
Data Analysis. The fluorescent gel data were collected
during the run by online laser detection. The obtained results
were analyzed using Gene Scan Analysis Software. For each
fluorescent fragment the size, height and area of the peak was
determined.
Calculation of Allele Ratios. In case of heterozygosity,
peak areas of the two alleles—in paired blood and tumor samples—were calculated by Gene Scan program. For the detection
of LOH, we required that one allele from the tumor sample was
reduced by more than 50%.
Stage and Grade. Stage was assessed prospectively with
a modification of the TNM system; the distinction between
muscle invasive tumors was based on tumor palpation before
transurethral resection (25). Ta tumors were confined to the
mucosa, T1 to the submucosa, muscle-invasive tumors without a
palpable mass were regarded as T2; a mobile palpable mass
4
Genome database: http//gdbwww.gdb.org.
Table 1 Background of patients
Characteristics
Tumor stage
Ta
T1
T2
T3
T4a
T4b
TX
Tumor grade
G1
G2A
G2B
G3
G4
GX
n (%)
122 (51.7)
35 (14.8)
34 (14.4)
13 (5.5)
2 (0.8)
2 (0.8)
28 (11.8)
13 (5.5)
75 (31.8)
45 (19.1)
65 (27.5)
5 (2.1)
33 (14.0)
defined T3 tumors; a mass judged to be invading the prostate,
rectum, vagina, or uterus categorized the tumor as T4a and
fixation to the pelvic wall as T4b. Grading was done with a
modification of the WHO system, which distinguishes grade 2A
tumors from grade 2B ones according to the basic tissue order
(26).
Statistical Analysis. The ␹2 test for trend was used to
assess the correlation between the frequency of LOH and pathological information. The relation between the number of LOH
and pathological information was analyzed by Spearman’s rank
correlation test.
RESULTS
We examined 236 transitional cell carcinomas of the urinary bladder using seven microsatellite markers for chromosome 13 with tri- or tetranucleotide repeats.
Background. Table 1 shows some characteristics of the
bladder cancer patients.
Heterozygosities. Table 2 shows heterozygosity for the
observed proportions of cases with each marker. It ranged
between 0.68 and 0.87, which were close to the reported rates
from the Genome Database.4
Deletion Mapping of Chromosome 13. Table 3 shows
the prevalence of loss of heterozygosity in relation to stage. In
two regions LOH was prevalent; one was at 13q12.3–14.2
(D13S894). In Ta tumors, seven cases (Fig. 1: K181, S44, S66,
S154, S144, S192, K123) had LOH at 13q12.3–14.2 together
with allelic retention at 13q14.3. The second frequently changed
region was at 13q14.3, which is within the RB gene. Six tumors
(Fig. 2: K110, S27, K120, S30, H25, K146) in stage T2 had lost
a single locus at 13q14.3, but the deleted loci comprised less
than three alleles alongside each other.
LOH and Replication Error on Chromosome 13. Of
the 236 tumors, 131 (56%) had LOH at 1 or more loci on
chromosome 13 and 17 (4%) had a replication error. LOH was
detected in 36 (21%) of 168 informative cases by D13S787, 41
(25%) of 161 by D13S894, 45 (22%) of 202 by D13S325, 54
(32%) of 170 by RB1.20, 41 (24%) of 173 by D13S800, 39
(26%) of 151 by D13S793, and 35 (22%) of 161 by D13S779
(data not shown).
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612 RB and Bladder Cancer
Table 2
Marker
1
2
3
4
5
6
7
a
D13S787
D13S894
D13S325
RB1.20
D13S800
D13S793
D13S779
Observed and reported heterozygosity at different loci in 13q
Locus
Observed heterozygosity
Reported heterozygositya
13q11–12.1
13q12.3–14.2
13q14.1–14.2
13q14.3
13q21.2–22
13q31–32
13q32
0.72 (169/235)
0.71 (167/235)
0.87 (203/234)
0.73 (173/236)
0.74 (175/236)
0.68 (161/236)
0.73 (163/232)
0.72
0.64
0.8
0.94
0.75
0.77
0.71
Data from the Genome Database 19981106 (Ref. 26).
Table 3
Prevalence of loss of heterozygosity at different loci in 13q in relation to tumor stagea
Locus
Ta (n ⫽ 122)
T1 (n ⫽ 35)
13q11–12.1
13q12.3–14.2
13q14.1–14.2
13q14.3
13q21.2–22
13q31–32
13q32
At least one
15/87 (17)
18/85 (21)
24/106 (23)
18/87 (21)
19/89 (21)
19/84 (23)
16/82 (20)
62/122 (51%)
6/23 (26)
9/24 (38)
8/31 (26)
8/25 (32)
10/26 (39)
8/22 (36)
6/23 (26)
20/35 (57%)
a
b
T2 (n ⫽ 34)
5/26 (19)
9/22 (41)
7/28 (25)
15/25 (60)
4/25 (16)
7/18 (39)
7/26 (27)
23/34 (68%)
T3 (n ⫽ 13)
T4a, T4b (n ⫽ 4)
Pb
2/12 (17)
1/10 (10)
2/13 (15)
5/9 (56)
1/9 (11)
0/8 (0)
1/11 (11)
6/13 (46%)
2/3 (67)
0/3 (0)
0/2 (0)
2/3 (67)
1/3 (33)
1/2 (50)
1/3 (33)
4/4 (100%)
0.273
0.105
0.872
0.001
0.264
0.154
0.701
0.146
Values in parentheses are percentages.
P based on ␹2 test for trend.
Fig. 1 Patterns of LOH on chromosome 13 in Ta bladder tumors.
LOH According to Tumor Stage and Grade at 13q14.3.
Table 3 shows the relation between LOH of each microsatellite
and tumor stage. In each marker, LOH was more prevalent for
higher stages, but a statistically significant trend was found for
13q14.3 only. The relation between LOH and tumor grade is
presented in Table 4. Again, a statistically significant trend was
found for 13q14.3 only. We also analyzed a collapsed variable:
LOH at least one of the seven regions included in the study. This
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Clinical Cancer Research 613
Fig. 2 Patterns of LOH on chromosome 13 in T1, 2, 3, 4a, and 4b bladder tumors.
Table 4
Prevalence of loss of heterozygosity at different loci in 13q in relation to tumor gradea
Locus
Grade 1 (n ⫽ 13)
Grade 2A (n ⫽ 75)
Grade 2B (n ⫽ 45)
Grade 3 (n ⫽ 65)
Grade 4 (n ⫽ 5)
Pb
13q11–12.1
13q12.3–14.2
13q14.1–14.2
13q14.3
13q21.2–22
13q31–32
13q32
At least one
0/12 (0)
1 /8 (13)
0/13 (0)
1/10 (10)
1 /8 (13)
1/11 (9)
1 /8 (13)
3/13 (23)
9/53 (17)
7/49 (14)
15/64 (23)
11/51 (22)
10/50 (20)
13/54 (24)
11/51 (22)
38/75 (51)
6/31 (19)
10/31 (32)
11/39 (28)
10/35 (29)
9/38 (24)
8/29 (28)
5/27 (19)
25/45 (56)
13/51 (26)
17/51 (33)
12/58 (21)
21/45 (47)
15/53 (28)
12/38 (32)
11/51 (22)
39/65 (60)
2/3 (67)
1/4 (25)
1/4 (25)
3/4 (75)
0/3 (0)
0/3 (0)
1/3 (33)
5/5 (100)
0.079
0.172
0.318
0.012
0.645
0.482
0.073
0.032
a
b
Values in parentheses are percentages.
P based on ␹2 test for trend.
variable was related to grade in a statistically significant way. In
grade 4, anaplastic tumors, 5 of 5 (100%) informative cases had
LOH at one or more loci.
A restricted analysis was done concerning RB; we selected the
cases that did not have any LOH at any of the six investigated loci
other than at 13q14.3. Among them, we documented the correlation
between LOH and tumor grade and stage, respectively. Four (8%)
of 41 Ta cases had the defined change, none (0%) of 11 cases with
T1 tumors, 5 (50%) of 10 with T2, 3 of 6 (50%) at T3, and 1 of 1
(100%) at T4. The corresponding figures for grade 1 was 1 of 9
(11%), grade 2A, 3 of 33 (9%), grade 2B, 0 of 14 (0%), grade 3, 7
of 22 (32%), and grade 4, 2 of 2 (100%). A statistically significant
relation was found with regard to tumor stage (P ⬍ 0.001) as well
as grade (P ⫽ 0.020; data not shown).
Number of LOH According to Tumor Stage and Grade
at Seven Investigated Loci. The relations between number
and percentage of LOH at seven investigated loci and tumor
stage and grade are presented in Table 5. We found a statistically significant relation with regard to tumor stage (P ⫽ 0.034)
as well as grade (P ⫽ 0.005) using Spearman’s rank correlation.
Fig. 3 shows the correlation between the average number of loci
with LOH and stage, and grade, respectively.
DISCUSSION
Our study adds further evidence to the notion that altered
RB function is important for the development and clinical behavior of some urinary bladder cancer cases. Clearly, there is a
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614 RB and Bladder Cancer
Table 5
Losses of heterozygosity at seven investigated loci in relation to stage and grade
Stageb
No. of LOH
Ta (n ⫽ 122)
T1 (n ⫽ 35)
T2 (n ⫽ 34)
T3 (n ⫽ 13)
T4a, T4b (n ⫽ 4)
0
1
2
3
4
5
6
7
62 (51)
31 (25)
11 (9)
9 (7)
6 (5)
1 (1)
2 (2)
0
15 (43)
4 (11)
6 (17)
4 (11)
4 (11)
1 (3)
1 (3)
0
11 (32)
9 (26)
4 (12)
5 (15)
3 (9)
2 (6)
0
0
7 (54)
4 (31)
1 (8)
0
0
0
1 (8)
0
0
2 (50)
1 (25)
1 (25)
0
0
0
0
Gradec
No. of LOH
1 (n ⫽ 13)
2A (n ⫽ 75)
2B (n ⫽ 45)
3 (n ⫽ 65)
4 (n ⫽ 5)
0
1
2
3
4
5
6
7
10 (77)
2 (15)
0
1 (8)
0
0
0
0
37 (49)
19 (25)
9 (12)
5 (7)
2 (3)
2 (3)
1 (1)
0
20 (44)
7 (16)
8 (18)
5 (11)
3 (7)
1 (2)
1 (2)
0
26 (40)
15 (23)
5 (8)
9 (14)
6 (9)
2 (3)
2 (3)
0
0
3 (60)
1 (20)
0
1 (20)
0
0
0
a
Values are number of occurrences, with percentage in parentheses.
P ⫽ 0.034; Spearman’s rank correlation test.
c
P ⫽ 0.005; Spearman’s rank correlation test.
b
Fig. 3 Average number of loss of hererozygosity at seven investigated
loci in relation to stage and grade.
relation between tumor grade and stage and the prevalence of
RB-related changes. We also showed, however, that inhibition
of RB alone is not sufficient to determine the tumor as aggressive. LOH at 13q14.3 was found in 11 of 59 (19%) tumors
classified as TaG2A or less, that is, in tumors that almost never
progress to being metastatic. Finally, we found an association
between grade and stage, respectively, and the number of loci at
13q with LOH. Which biological phenomenon this finding
reflects, if any, remains to be elucidated.
No previous study on bladder cancer has addressed associations with the variable “number of loci with LOH at 13q,”
and our findings must be substantiated before any biological
significance is looked for. The variable may simply be a marker
for genomic instability caused, in turn, by previous genetic
changes, e.g., by the genetic events that initially formed the
malignant cell. Nevertheless, it may also be true that nonspecific
loss of normal cellular function contributes to degree of malignancy. Our data, together with cytogenetic studies showing
deletions in a large number of areas outside of those harboring
p15, p16, p19, p53, and RB (1–5), may imply that altered
expression of several additional genes is important for urinary
bladder cancer growth.
Our study covered LOH within a broad area on 13q. We
did find LOH at all loci, and in 131 of 236 (56%) cases at least
one locus was measured as being deleted. These observations
cannot only be explained by frequent deletion of a large allele
including RB: we restricted an analysis to cases with no LOH at
13q14.3, and still 42% of the cases had LOH at at least one
locus. Previous evidence for deletion at 13q outside of RB has
been presented by Habuchi et al. (14). In 39 informative cases,
they found a prevalence of LOH of 30% at 13q22, of 25% at
13q31, and of 18% at 13q34. These figures correspond to ours.
An altered function of an unknown gene that is important for the
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Clinical Cancer Research 615
pathogenesis of the bladder tumor provides one explanation to
the data. The altered function also may just be random events
resulting from the malignant transformation.
In 170 informative cases, we found that the LOH prevalence of an allele within RB varies with stage and grade, respectively. Our findings corroborates those of Cairns et al. from 162
patients, of Knowles et al. analyzing 83 tumors and by
Miyamoto, based on 45 observations (4, 5, 13). The combined
evidence leaves no doubt that typically there is an association
between the possibility of a fatal course of a case with transitional cell carcinoma and an altered RB function. However, it is
also clear that LOH occurs at 13q14.3 in bladder tumors with a
low stage and grade. Cairns reported for Ta and T1 cases 2 LOH
in 48 tumors, for T2⫹, 26 LOH in 46 tumors, for grade 1, 2
LOH in 31 tumors, for grade 2, 8 LOH in 25 tumors, and for
grade 3, 18 LOH in 38 tumors (4). We can conclude that
inhibition of RB is not sufficient cause for a urinary bladder
tumor to be potentially lethal. Studying the suggested interaction
between RB and p16 in human tumors could possibly expand
our knowledge in this regard.
It is also clear from the combined data that inhibition of RB
is not a necessary cause for a transitional cell to become malignant. We could not detect LOH on the markers within RB in
68% of the informative cases. The corresponding figures for
Cairns are 70%, for Knowles, 85% and, for Miyamoto, 77% (4,
5, 13). Thus, reasonably at least two pathogenetic pathways
operate in the disease. Suggested models include one in which
inhibition of p53 is central and another with RB. As the evidence
accumulates concerning mutation, methylation, and LOH of, for
example, p16, p19arf, p53, p21, p27, and RB in relation to stage,
grade, and clinical course (for various treatment approaches), a
better understanding of the relevant genetic events for urinary
bladder cancer will probably be achieved. Unselected series
might possibly be important for the pursuit; we will continue to
analyze inhibition of tumor suppression genes in this population-based series.
ACKNOWLEDGMENTS
We thank Professor Katsusuke Naito for his continuous encouragement.
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Bladder Cancer: Allelic Deletions at and around the
Retinoblastoma Tumor Suppressor Gene in Relation to Stage
and Grade
Takashi Wada, Jari Louhelainen, Kari Hemminki, et al.
Clin Cancer Res 2000;6:610-615.
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