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JOURNAL OF PATHOLOGY, v o l .
179:
1 6 9 - 1 7 6 (1 9 9 6 )
LOSS OF CHROMOSOME 9 IN TISSUE SECTIONS OF
TRANSITIONAL CELL CARCINOMAS AS DETECTED
BY INTERPHASE CYTOGENETICS. A COMPARISON
WITH RFLP ANALYSIS
PINO J. PODDIGHE*!, PIERRE-PAUL BRINGUIERÎ, MONIQUE VALLINGA*, JACK A. SCHALKENj, FRANS C. S. RAMAEKERS*
AND ANTON H. N. HOPMAN*
* Department o f Molecular Cell Biology and Genetics, University o f Limburg, Maastricht, The Netherlands; Departments o f
t Pathology and. J Urology, University Hospital Nijmegen, The Netherlands
SUMMARY
Interphase cytogenetics by in situ hybridization (ISH) using a panel of centromere-associated DNA probes for chromosomes 1, 7, 9,
10,11,16,17, and 18 was performed on 5 /mi thick frozen tissue sections of transitional cell carcinomas (TCCs) of the urinary bladder.
By this approach, chromosome ploidy, numerical chromosome aberrations, imbalance between chromosomes, and heterogeneity of
aberrations within individual tumours were determined. In 15 of 24 TCCs, loss or underrepresentation of chromosome 9, compared with
the ISH copy numbers of at least five other chromosomes, was demonstrated. Independently, RFLP analysis were performed on the same
cases to detect loss of heterozygosity (LOH) of chromosome loci 9q34, llp lS , 16q22-24,17pl3, and 18q21. LOH was found in 9 of 19
informative cases for chromosome locus 9q34. Comparison of the ISH and RFLP results showed no correlation between numerical
aberration and LOH for the loci on chromosomes 11,16,17, and 18. However, numerical loss of chromosome 9 was found in 89 per cent
(eight of nine cases) with LOH for 9q34. Conversely, LOH at 9q34 was observed in only 67 per cent (eight of 12 cases) with
underrepresentation of chromosome 9. Moreover, in 60 per cent of the non-informative cases (three of five cases), underrepresentation
for chromosome 9 was observed. These results indicate that the heterochromatin probe for chromosome 9 can be reliably used in TCC
tissue sections for the detection of chromosomal loss. In aneuploid TCCs, this DNA probe can be used for the detection of chromosomal
underrepresentation only in combination with other centromere-associated DNA probes.
KEY WORDS—bladder cancer; chromosome aberrations; in situ hybridization; RFLP analysis
INTRODUCTION
Transitional cell carcinoma (TCC) is the most
common form of urinary bladder cancer, comprising a
heterogeneous group with markedly different neoplastic
features.1 Approximately two-thirds o f patients with
TCC sooner or later present recurrences, o f which 10-25
per cent will be of higher grade or stage.2 Currently, the
most important predictors of the behaviour o f TCCs are
histological stage and grade. The D N A ploidy, as deter
mined by flow cytometry (FCM), may have additional
value in prediction of the biological behaviour o f the
tumour.3 For example, FCM has revealed that noninvasive (pTa) TCCs are predominantly DNA-diploid,
while invasive behaviour of these malignancies is highly
correlated with D N A aneuploidy or tetraploidy.4“6
Cytogenetic analysis by karyotyping o f TCCs has
revealed several specific chromosomal changes, such as
—9 or —9q —. 1
Comparison of tumour D N A and constitutional
D N A for polymorphic markers by restriction fragment
length polymorphism (RFLP) analysis allows the detec
tion of loss of heterozygosity (LOH) at specific chromo
somal loci.12 RFLP analyses have shown that allelic loss
Addressee for correspondence: Pino J. Poddighe, Department of
Pathology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB
Nijmegen, The Netherlands.
CCC 0022-3417/96/060169-08
© 1996 by John Wiley & Sons, Ltd/
on 9q, l i p, 16q, 17p, and 18q is involved in various
human cancers, including urinary bladder cancers.13“18
The RFLP probe for 9q (9q34 locus), in particular, has
been shown to be indicative for changes in chromosome
9 in bladder cancer. The existence o f two allelic loci has
been postulated, one in each o f both chromosomal
arms.19 Recently, the deleted regions on chromosome
9 in bladder cancer were better defined.20 It has
been suggested that 9p LOH may be associated with a
more aggressive biological behaviour or early disease
progression.20
Interphase cytogenetics by in situ hybridization (ISH)
using centromere-associated D N A probes is applied to
both single cell suspensions and sections o f frozen tissues
or formalin-fixed, paraffin-embedded tissues.21-30 In this
study, interphase cytogenetic analysis was performed to
detect numerical chromosomal aberrations in standard
5 jam thick frozen tissue sections. Selection of the probes
was made on basis of the literature, indicating specific
loss for chromosomes 9, 10, and 11, or gain for chromo
somes 1 and 7.31»32 In addition, centromeric probes for
chromosomes 16, 17, and 18 were selected, of which no
numerical changes have been reported. W e used several
RFLP probes on the p- or q-arms o f these chromosomes
to detect LOH in the D N A extracted from serial sections
o f the same samples.
Although the D N A probes for ISH recognize
repetitive sequences in the centromeric regions of
Received 1 December 1994
Accepted 22 January ¡996
P. J. PODDIGHE ET AL.
170
chromosomes and the RFLP probes were used to detect
LOH on the p- or q-arms, we focused the comparison
on the following questions: (a) is there a correlation
between underrepresentation of certain chromosomes,
as detected by ISH, and LOH of markers on that same
chromosome? (b) Can heterogeneity of chromosome
aberrations be detected in TCC by means of ISH and
how does such heterogeneity influence the RFLP results?
(c) To what extent do both techniques overlap and thus
reveal the same information?
MATERIALS AND METHODS
Sample selection and preparation for ISH
The tissue samples were selected for their tumour cell
content, which was at least 60-70 per cent. Classification
of histological stage and grade (I—III) was performed
using paraffin sections. For this study, 24 frozen tissue
samples o f TCC were selected, on which both ISH and
RFLP techniques could be performed. For ISH, 5/im
sections were mounted on organosilane-coated slides,
air-dried, and stored at room temperature. For RFLP
analysis, 20
sections were collected and stored at
— 20°C. Also, peripheral blood lymphocytes (PBLs)
were collected from the same patients for RFLP
analysis.
Pretreatment o f frozen sections
Prior to the ISH procedure, the sections were fixed in
70 per cent ethanol/1 per cent formaldehyde for 20 min
at -2 0 °C . After subsequent washing in phosphatebuffered saline (PBS)/0*05 per cent Tween-20, and water
for 5 min at room temperature, the slides were incubated
for 15 min at 37°C in 100 /¿g pepsin per ml 0*01 N HCL
Then the slides were rinsed in five dip washes of water
and five dip washes o f PBS, and dehydrated in an
ascending alcohol series. After air-drying, the sections
were post-fixed in 1 per cent formaldehyde/PBS for 10
min at room temperature.
DNA probes for ISH
The centromere-associated D N A probes pUC1.77,33
p 7t.l,34 pHUR98,35 pHUR195,35 plO .l,36 p L C llA ,37
pl7H 8, and LL8439 were used for the detection of
target sequences on chromosomes 1, 7, 9, 16, 10, 11, 17,
and 18, respectively. The probes were labelled by nicktranslation with biotin-11-dATP, according to the sup
plier's labelling kit instructions (Boehringer, Mannheim,
Germany).
In situ hybridization
The D N A probes described above were hybridized to
the (tumour-) cell preparations at a probe concentration
of 1 ngljul hybridization mixture, containing 60 per cent
formainide-2 x standard sodium citrate (SSC)-IO per
cent dextran sulphate, pH 7*0. Under these stringent
conditions, 15/d of the hybridization mixture was
applied to the slides under a coverslip (18 x 18 mm).
Dénaturation of probe and target D N A was performed
simultaneously by heating the slides in a moist chamber
to 70°C for 3 min. After hybridization for 2-16 h at
37°C, the coverslips were removed by immersing the
slides in 2 x SSC, pH 7-0 at 42°C. Post-hybridization
washing steps were performed twice in 60 per cent
formamide-2 x SSC, pH 7*0 for 5 min and twice in
2 x SSC, pH 7*0 for 5 min at 42°C.
Immunocytochemical detection o f the hybridized
DNA probes was performed as previously described24,25
with mouse anti-biotin (DAKO A/S, Glostrup,
Denmark), biotin-labelled horse anti-mouse IgG
(Vector, Burlingame, CA, U.S.A.), and a final incuba
tion with the avidin-biotin-labelled peroxidase complex
(Vectastain Elite ABC Kit, Vector). All immuno
cytochemical steps were performed for 30 min at 37°C.
The D N A probe was visualized with 0*5 mg/ml 3,3'diaminobenzidine tetrahydrochloride (DAB, Sigma),
0*65 per cent imidazole (Marck, Darmstadt, Germany),
0*015 per cent H20 2 (Merck) in PBS for 3 min at pH 7*8,
and the signal was amplified with C uS04 (0-5 per cent in
0*9 per cent NaCl) for 3 min. The slides were counterstained with haematoxylin and mounted in Permount
(Fisher Scientific, New Jersey, U.S.A.).
Evaluation o f ISH results
The reproducibility and validity of a protocol to
detect and evaluate chromosome copy numbers by ISH
in 5/im routinely processed tissue sections have been
described elsewhere.25
A disadvantage of the use o f tissue sections is that due
to truncation of a significant number of nuclei, the
number o f ISH signals per nucleus will be under
estimated compared with the actual copy number o f the
target chromosome.25“28 Therefore, we evaluated sepa
rately within each section the tumour area, normal
tissue, and stromal cells, since no simple correction
factors for truncation of nuclei are available so far. In
diploid tumours, the true chromosome copy number is
detectable in about 50 per cent of the nuclei, so that
monosomy or trisomy can be determined conclusively.
However, with increasing nuclear size, the true chromo
some copy number will be increasingly underestimated.
For this reason, hybridization with different DNA
probes on the same tumour areas in parallel sections
is necessary to study the imbalance of chromosome
copy numbers and, as a result, the specific loss of
chromosomes.
Statistical methods
4
The Kolmogorov-Smirnov test was used, Under
representation of a specific chromosome was seen as a
shift to the left of the ISH signal distribution when
compared with non-aberrant probe distributions. Con
versely, gain of a specific chromosome was seen as a shift
to the left. P values of 0-001 or less were considered
significant.
DNA isolation and Southern blot analysis
RFLP analysis was performed as described previ
ously.40 Briefly, high molecular weight DNA (about
171
ISH AND RFLP ANALYSIS OF BLADDER CANCER
Table I— A sum m ary o f the R F L P and ISH results for the 24 transitional cell carcinomas. For ISH, the
centromere-associated D N A probes for chrom osom es 1, 7, 9, 10, 11, 16, 17, and 18 were used
R F L P analysis
Case
No.
G rade/stage
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
G 1/pTa
G l/p T a
G l/p T a
G 1/pTa
G l-2 /p T a
G l-2 /p T a
G 2/pT a
G 2/pT 1
G 2 /p T l
G 2 /p T l
G 2/pT 1
G3/pT3
G3/pT3
G 2 -3 /p T a
G 2/pT 1
G 2/pT 1
G 2/pT 1
G l-2 /p T l-2
G 3/pT2
G 3 /p T > 2
G3/pT3
G 1-2 /p T a
23
24
•
C hrom osom al locus detected on
9q
lip
16q
17p
1Sq
+
+
—
+
+
—
+
—
—
+
+
—
NI
NI
+
+
+
+
+
—
NI
+
—
NI
+
«wjw
+
+
—
NI
i
+
NI
NI
PI»||
—
NI
+
—
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
—
+
+
+
NI
NT
+
+
—
+
+
------
NI
NI
+
ll i 1É
+
+
------
H"
+
G 1~2/pT a
G 3 /p T > 2
ISH analysis
+
+
NI
+
NI
+
+
—
NI
NI
NI
nd
nd
NI
+
—
—
+
NI
NI
nd
nd
+
+
m = probe is not informative; nd^-not done; + =no LOH;
25 /¿g) was isolated from frozen tissue samples of the 24
TCCs and from whole blood samples of the same
patients as described.41 Ten /zg of D N A was digested
with Taql of Mspl and separated on a 0*8 per cent
agarose gel and transferred to Hybond N + (Amersham)
according to the manufacturer’s instructions. The 32P
dATP-labelled probes, EFD126 for 9q34,42 c-H-ras for
U p l 5 43 PV962 and 79.2.23 for 16q22,44,45 144D6 for
17pl3,46 and 15.65 for 18q21,47 were hybridized over
night in 250 mM N a2H P 0 4, 1 m M EDTA, 1 per cent
BSA, 7 per cent SDS, pH 7*2 at 65°C Blots were then
washed successively in 250 mM N a2H P 04, 1 m M EDTA,
1 per cent SDS, pH 7*2; in 125 mM Na2H P 0 4, I m M
EDTA, 1 per cent SDS, pH 7*2; and in 50 mM N a2ITP04,
1 m M EDTA, 1 per cent SDS, pH 7-2 40 Since tumour
content was high enough, scanning of film was not
necessary for interpretation. The RFLP analysis was
performed blind and independently of the ISH analysis.
RESULTS
Table I summarizes the RFLP and ISH results for
each of the 24 TCCs. These were ranged according to
their chromosome ploidy as determined on basis of ISH.
C hrom osom e
ploidy
Numerical
aberration(s)
Diploid
Diploid
Diploid
Diploid
Diploid
Diploid
Diploid
Diploid
Diploid
Diploid
Diploid
Diploid
Diploid
Triploid
Triploid
Tetraploid
Hexaploid
Tetraploid
Tetraploid
Tetraploid
Triploid
Tetraploid
/diploid
Tetraploid
/diploid
Tetraploid
/diploid
N one
- 9 , - 17
-9
- 9 , - 17
-1 0
—9
- 9 , - 18
—9
+ 7, - 9
- 18
None
-9
None
-9
+1, —9
+ 1, - 9
-9 , -9
None
- 18
-9 ,
17
N one
+ 1,
9,
9
/+ 1 ,
9
None
-
-
—
—
- 9 , - 9 , - 17, ~ 17
/ - 9, - 17
ni« 4
Cases 1-13 were classified as chromosome diploid and
cases 14-24 as aneuploid, of which cases 22-24 were also
heterogeneous, i.e., mixed diploid and aneuploid.
Evaluation of the ISH reactions showed in 18 patients
27 times a loss or gain of ISH signals compared with the
mean chromosome ploidy. A numerical loss of chromo
some 9 was found in 15 cases (62-5 per cent). In 13
patients, we found 24 times LOH for one or more
markers. The most frequent allelic loss was seen for the
marker on chromosome 9q. Of 19 informative cases (79
per cent), we found LOH of the 9q34 locus in nine
tumours (47 per cent).
Chromosome diploid tumours ( cases 1-13)
Numerical chromosome aberrations were detected in
10 ont of 13 diploid TCCs (Table I). In eight cases, a loss
of chromosome 9 was observed. In two cases, an ad
ditional loss of chromosome 17 was detected. One case
(case 4) demonstrated a loss of chromosome 10, whereas
case 6 showed loss of chromosome 18, next to a loss of
chromosome 9. Loss of chromosome 18 was also
observed in case 9. Figure 1A shows the quantitative
evaluation of the hybridization signals of case 1 for the
centromere-associated D N A probes for chromosomes 1,
V. J. P0DD1GHE ET AL.
172
% o f nuclei
80
70
60
50
C
A
-
40
-
50
-
40
-
30
% of nuclei
30
-
I
20
i1 1 '
-
10
fill
life
I
III
-
0
o
T
1
I
-
ill
I■li»
20-
ili'
10 -
illy
i
3
h
T
1
0
ill»]
iiiij
i
2
T
4
Mm
ill
I
a_l I
0i l
I
80
70
*"
50
-
40
-
30
-
;
S S rii
li
» S il
■ S S ítf
n
m.
T
6
5
3
% of nuclei
D
B
1
40
-
30
-
I
«i-
i
2010 0
I
number o f ISH signals per nucleus
50
-
60
I
ilu
T
number o f ISH signals per nucleus
% of nuclei
I,
I
«
L i*
0
È
III“
'
III'
fc y
I
IÌ '
20-
1
m
m
10 -
àil
is
le
m
■ I
i
0
3
number o f ISH signals per nucleus
T
0
T
2
1
m
M;
8
number o f ISH signals per nucleus
Fig. 1— Frequency distribution patterns of ISH signals obtained with different centromere-associated D N A probes for chromosome 1 (■),
chromosome 9 (H), chromosome 11 (□ ), chromosome 17 (H), and chromosome 18 (H) on frozen tissue sections of TOCs. (A) Case 1, representing
a diploid tumour with no numerical aberrations. (B) Case 3, showing underrepresentation for chromosome 9, (C) Aneuploid tumour, case 16,
demonstrating overrepresentation of chromosome centromere 1 and loss of the centromeric target of chromosome 9. (D) Case 17, representing a
highly aneuploid tumour with loss o f chromosome 9. The mean chromosome copy number is 6. Note the effect of truncation of nuclei on the
distribution patterns of the chromosome copy numbers
9, 11, 17, and 18. In this particular case, the mean
number of ISH signals for the chromosomal probes was
two in about 45-50 per cent o f the nuclei and one in
2 8 ^ 2 per cent o f the nuclei. These percentages did not
differ from those obtained in model studies.26,48 This
case can therefore be classified as a chromosome diploid
tumour. Furthermore, statistical analysis revealed no
differences in the ISH signal distribution pattern of the
chromosomes. Figure IB represents case 3, in which a
significant loss of chromosome 9 was detected (with all
investigated chromosomes, P < 0 ’001)s since 68 per cent
o f the nuclei contain only one ISH signal for the
chromosome 9 probe. In about 55 per cent of the nuclei
o f this case, two ISH signals for the other chromosomes
were detected. Figures 2A and 2B show tumour areas of
this chromosome diploid case (case 3) in frozen tissue
sections, hybridized with the probe for chromosome 11
(Fig. 2A), showing two ISH signals, and the probe for
chromosome 9 (Fig. 2B), showing only a few cells
containing two ISH signals per nucleus, indicating an
evident monosomy for chromosome 9.
The RFLP results for cases 1 and 3 are shown in Figs
2C and 2D, on which Taql-digested D N A is hybridized
to a probe for 9q and l i p, respectively. Tumour DN A
from case 3 showed LOH for the 9q probe (Fig. 2C),
whereas for the l i p probe no LOH was observed
(Fig. 2D).
Chromosome aneuploid tumours (cases 14-24)
In 5 of 11 aneuploid cases, underrepresentation of
chromosome 9, compared with the other chromosomes,
was detected. In two cases (cases 15 and 16), overrepre
sentation of chromosome 1 was observed. An additional
loss of chromosome 17, next to loss o f chromosome 9,
was detected in case 20. Case 19 showed loss o f chromo
some 18 as the only numerical aberration.
Figure 1C shows the frequency distribution of case 16
by evaluating the ISH signals in 200 nuclei in serial
sections, showing a mean between 3-0 and 3-3 for the
copy number of chromosomes 7, 11, 16, 17, and 18, and
a modal copy number of four ISH signals per nucleus.
For chromosome 1 the mean copy number was 3-9, and
the modal copy number was five to six ISH signals per
nucleus, representing a relative overrepresentation or
gain of this chromosome (P values<0*001). The mean
and modal copy numbers for chromosome 9 were 2*4
and 2, respectively, demonstrating an underrepresen
tation or loss of this chromosome (P values<0-001).
The frequency distribution of ISH signals for chromo
somes 1,9, 11, 17, and 18 o f case 17 is shown in Fig. ID.
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P. J. PODDIGHE E T A L
174
The mean copy number for chromosomes 1, 11, 17, and
18 was between 5-6 and 5-9, and there was a modal copy
number of six ISH signals per nucleus. For chromosome
9 the mean copy number was 3*6, with a modal copy
number of 4, representing a loss of two copies of this
chromosome (P values<0*001). Figures 2E and 2F rep
resent part of the tumour of case 17, where the nuclei
contain five to six ISH signals, with a maximum ISH
copy number of 9, for chromosome 1 (Fig. 2E), whereas
for chromosome 9 the nuclei contain three to four
ISH signals, with a maximum ISH copy number of 6
(Fig. 2F).
Southern blots o f tumour and constitutional DNA
from cases 16 and 17 are shown in Figures 2G and 2H,
hybridized to the 9q and 1 lp probe, respectively. For the
9q probe, case 16 demonstrates LOH, while case 17 is
not informative (Fig, 2G). For the lip probe, case 16
retains the lip allele, whereas case 17 reveals LOH
(Fig. 2H).^
Evaluation of ISH signals for the three aneuploid
heterogeneous cases (cases 22-24) revealed extensive
chromosome heterogeneity, both in the range of ISH
signals for the eight different D N A probes in the indi
vidual tumour cells and between different tumour areas
in the same case o f TCC. We have therefore classified
these three cases as heterogeneous. In two cases (cases 22
and 24), loss of the heterochromatin region of chromo
some 9 was detected after screening of serial sections
with the eight centromere-associated D N A probes. In
the tetraploid tumour areas, we observed an ISH copy
number of chromosome 9 of maximum two indicated as
an apparent loss o f two copies o f chromosome 9 (Table
1). Figure 21 demonstrates the tumour heterogeneity of
ISH signals in case 24, in which one part of the TCC
contains three or four ISH signals for the chromosome 1
probe, while in another part o f the TCC mainly one or
two ISH signals for the same probe are found.
Figures 2 S and 2K demonstrate the RFLP results of
D N A from this tumour hybridized to the 9q and lip
probes. Although LOH for the 9q locus was concluded,
the heterogeneity of the tumour D N A is possibly
reflected by the faint band in the blot (arrow in Fig. 2J).
DISCUSSION
Several investigations have demonstrated that
centromere-associated D N A probes can be applied in
ISH techniques to determine numerical chromosome
aberrations in interphase nuclei o f tumours.21-30
Thompson ei aL49 described a method o f hybridization
to sections more than 20 jam thick, to overcome difficul
ties in the analysis o f gene or chromosome copy number
due to truncation of nuclei. Since these analyses have to
be performed using laser-scanning confocal microscopy,
this approach is not routinely applicable.
Our study shows the application of ISH using a panel
o f D N A probes to standard 5 ¡am thick frozen tissue
sections of TCCs, in order to detect numerical chromo
some aberrations and chromosomal imbalances. The
most characteristic numerical aberration detected by
ISH in the 24 TCCs was a loss o f chromosome 9 in 15 of
24 cases (62-5 per cent; P valuescO-OOl), compared with
the mean chromosome ploidy determined with the panel
of eight centromere-associated D N A probes. These
observations strongly confirm other published data,
where the cytogenetic observations concerning loss of
chromosome 9 in low-grade, low-stage TCCs were con
firmed by the ISH approach. Moreover, the frequent
occurrence of loss of chromosome 9 in early stages of
TCC is in agreement with the observation of Tsai et
a/.,33 who showed that LOH of markers on chromosome
9q could be detected in 67 per cent of the informative
cases. Comparison of the ISH results with RFLP analy
sis revealed that numerical loss of chromosome 9 was
found in 89 per cent of the cases with LOH for 9q34.
However, only 67 per cent o f cases with loss of chromo
some 9 as detected by ISH showed LOH for 9q34.
The process of tetraploidization is a generally
accepted concept in tumour progression.12 In the aneu
ploid TCCs, the apparent loss of two copies of chromo
some 9 strongly indicates that the loss of one copy of
this chromosome occurred before tetraploidization took
place. This confirms our previous suggestion that loss of
chromosome 9 is a primary or early event in carcino
genesis of the urinary bladder31 and that it is conserved
during the process of tumour progression and invasion.
The loss of chromosome 9 as detected by ISH is most
probably not limited to a small part of 9q. Because this
D N A target is situated in the heterochromatin on 9q,
close to the centromere, only a complete loss of this
heterochromatin region would result in a complete dis
appearance of the chromosome 9 signal. Deletion of
either 9q or 9p would preserve part of this target
sequence, resulting in positive but less intense or less
extended ISH signals. In our RFLP analysis we used one
polymorphic marker located at 9q34. This may explain
our observation that in four cases (2, 4, 7, and 15) there
was an ISH loss of chromosome 9, but no LOH was
observed with RFLP analysis. These cases should
involve complex chromosomal rearrangements, with
loss of the he ter o chromatin region of chromosome 9 and
retention of the 9q34 region. Translocation of part o f the
9q-arm could be the most likely explanation.
For the other chromosomes studied, we found no
correlation between LOH and centromeric loss. The
biological meaning of these observations could be that
in those cases where LOH was found but no loss was
observed by ISH for that particular chromosome, an
interstitial deletion, chromosomal rearrangement, or
duplication of a part of that chromosome was involved.
Putative tumour suppressor genes involved in bladder
carcinoma are located at the region between 9pl 2—13
and 9q34.20,51,52 The loss o f the putative tumour sup
pressor gene on chromosome 9 also comprises, in most
cases, the heterochromatin region on 9q. ISH with a
centromere 9 probe can therefore be used to monitor
this loss in pathological specimens. On the other hand,
loss of tumour suppressor genes on chromosomes l ip
and 17p, which is frequent in bladder tumours, is not
associated with the loss of any entire chromosome or
with partial loss including the centromere. Screening for
loss of these tumour suppressor genes by ISH should
be done using specific cosmid probes located near the
^
O 1
'I ^
ISH AND R FL P ANALYSIS O F BLADDER CANCER
relevant locus. In this respect, the strategies of
Matsumura et al.53 demonstrated that LOH of 17p in
breast tumours correlated with loss of ISH signals of a
17p cosmid probe. N o correlation was found with the
centromeric probe. Similarly in our studies on bladder
tumours, no correlation was found with the copy
number for the centromeric probe.
ISH analysis using tissue sections is a more specific
approach than RFLP to karyotyping, since it is possible
to evaluate results at the individual cell level, while
Southern blot analysis provides a quantitative but in
tegral overview of changes that dominate within the
tumour. In our study, in three aneuploid cases in which
LOH for the chromosome 9 locus was found, we
detected a DNA diploid tumour area next to the D N A
aneuploid area. An ISH approach can discriminate
between these. Tissue microdissection and subsequent
PCR-RFLP analysis will allow the examination of LOH
o f specific chromosomal regions.54,55 Different clones
that might exist among neoplastic cells can then be
determined and studied in greater detail.
ACKNOWLEDGEMENTS
We wish to thank Dr C. van de Kaa for revision of the
pathological classification. H. Robben is acknowledged
for her assistance in the screening and evaluation of the
ISH results. Dr M. M. M. Pahlplatz and Dr P. C. M.
De Wilde are acknowledged for their help in statistical
analysis. This work was supported by The Netherlands
Cancer Foundation, project No. IKL 88-07.
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